Oral Health Improving Compositions

ABSTRACT

The present invention is concerned with microorganisms or fragments thereof as sensorically neutral oral care agents, particularly for prevention of dental calculus, as anti-caries agents and/or anti-oral malodor agents. The invention is furthermore concerned with compositions comprising microorganisms or fragments thereof for reducing mutans Streptococci. Such compositions can be used in oral care compositions, e.g. for caries prophylaxis, or for prophylaxis of dental calculus or oral malodor. They may also or instead be used for prevention or treatment of oral malodor. As the microorganisms and fragments thereof according to the present invention have a very low, unobtrusive smell and taste, they are particularly suited as sensorically neutral agents for preventment of dental calculus, caries, oral biofilm formation and/or for prevention or treatment of oral malodor. The microorganisms and fragments thereof, and also compositions comprising such microorganisms and fragments, can thus advantageously be used in food and feed compositions, particularly in pet foods. Furthermore, the invention is concerned with methods of preparing such microorganisms, fragments, compositions, foods and feeds.

The present invention is concerned with microorganisms or fragmentsthereof as sensorically neutral oral care agents, particularly forprevention of dental calculus, as anti-caries agents and/or anti-oralmalodor agents. The invention is furthermore concerned with compositionscomprising microorganisms or fragments thereof for reducing mutansStreptococci. Such compositions can be used in oral care compositions,e.g. for caries prophylaxis, or for prophylaxis of dental calculus ororal malodor. They may also or instead be used for prevention ortreatment of oral malodor. As the microorganisms and fragments thereofaccording to the present invention have a very low, unobtrusive smelland taste, they are particularly suited as sensorically neutral agentsfor preventment of dental calculus, caries, oral biofilm formationand/or for prevention or treatment of oral malodor. The microorganismsand fragments thereof, and also compositions comprising suchmicroorganisms and fragments, can thus advantageously be used in foodand feed compositions, particularly in pet foods. Furthermore, theinvention is concerned with methods of preparing such microorganisms,fragments, compositions, foods and feeds.

One of the notorious problems of tooth-bearing animals is the decay ofsuch teeth. This problem is of particular importance to such animalsthat cannot, like sharks, shed their teeth to continuously replace themby a new set. Among the most important causes for tooth decay is caries.By the action of microorganisms colonizing the oral cavity dental enamelis continuously weakened and ultimately dissolved, leading to theformation of cariotic lesions. Such lesions in turn are suitable forfurther colonization of microorganisms, aggravating the problem ofcaries.

A problem unrelated to the formation of caries is the development oforal malodor. Oral malodor is considered the result of unwantedmicroorganisms colonizing the oral cavity. However, no singlemicroorganism has so far been indicated as the primary cause for oralmalodor. It seems to be clear, however, that the formation of caries andof oral malodor are independent processes, as oral malodor can alsooccur in the absence of teeth, and caries can occur in the absence oforal malodor.

Another problem of oral health particularly in animals is the formationof dental calculus. Calcified deposits on teeth are formed bymicroorganisms colonizing the surfaces of teeth. Typically, within 7-10days calcified deposits on teeth have grown to such extent that they arevisible to the naked eye. Dental calculus forms a focus to allowcolonization of teeth by further microorganisms. This, in turn, can leadto the development of oral malodor, caries and inflammations of gingivaltissue. There is thus a need for the prevention of the development ofdental calculus, e.g. by slowing down dental calculus formation.

It has therefore frequently be tried to ameliorate these problemsindependently. Among the most notable solutions so far common in the artare tooth pastes and mouth washes. Such tooth pastes and mouth washesgenerally comprise antimicrobially effective agents to reduce the numberof or inhibit the activity of tooth colonizing microorganisms,particularly caries causative microorganisms and/or oral malodorgenerating microorganisms. However, due to the uncorrelatedness ofcaries and oral malodor formation, many agents effective against cariesare ineffective for prevention or treatment of oral malodor, and viceversa. Also, efficiency against dental calculus formation needs to beimproved.

Another problem is that antimicrobially effective substances frequentlyexhibit a strong intrinsic smell or taste, which is considered repulsiveby consumers and particularly by pet animals. Thus, compositions fororal care frequently comprise further olfactorily active agents to maskor alter the taste or smell inherent in the antimicrobially effectivesubstances. However, such further agents are also not readily acceptedby consumers due to their even stronger intrinsic taste or smell. Thisresults in an undesirably low compliance of consumers to the applicationrecommendations of manufacturers of such oral care compositions.

The problem of compliance is particularly important with children andanimals. Both are prone to devising cunning ways of avoiding oral carecompositions or of misusing them, e.g. by swallowing, such that anantimicrobially effective agent or agents cannot fully exert theirintended action. Resistance particularly of pet animals like cats anddogs to any form of medication is frequently lamented. Also, suchanimals are known to avoid medications including antimicrobiallyeffective oral care compositions even when hidden in a feed they wouldotherwise accept. In addition certain modes of applications for oralcare compositions are not available for animals, as they for example areunable to gargle. Also, certain treatments of the oral cavity areparticularly stressful to animals and would require anesthesis. Forexample, animals like cats and dogs will furiously resist removal ofdental calculus.

In summary, oral hygiene is considered tedious for human beingsincluding children and for animals including pet animals alike. Thepresent invention therefore intends to ameliorate the above problems ofprior art. In particular the present invention aims at increasingcompliance with oral care instructions for human beings and for animalsand particularly pet animals.

According to the invention, it is important for furthering compliancethat the agent or agents used for prevention of dental calculus, cariesand/or for prevention or treatment of oral malodor has a low intrinsictaste and scent. Thus, instead of masking or altering the taste andsmell such agents by addition of further strongly tasting or smellingagents the present invention is concerned with a different approach. Theinherent taste and smell of oral care agents had so far being overlookedor not been considered to be of major importance. Furthermore, it hasnow surprisingly turned out that agents are available which are botheffective for prevention of dental calculus, caries and/or prevention ortherapy of oral malodor without having a strong inherent taste or smell.It has also surprisingly been found that such agents are useful toincrease compliance with oral care instructions and particularly toreduce oral care avoidance behavior in humans, including children, andanimals, including pet animals. Also, the present invention allows toprepare oral care compositions having other flavors or fragrances thanpreviously required for masking or altering the taste or smell ofanti-calculus agents, anti-caries agents and/or anti-oral-malodoragents.

The present invention is concerned with microorganisms or fragmentsthereof as sensorically neutral oral care agents. The invention isfurthermore concerned with compositions comprising microorganisms orfragments thereof for reducing mutans Streptococci. Such microorganismsand fragments thereof, and also such compositions, can be used fortreatment or prevention of dental calculus. Such microorganisms,fragments or compositions can also be used in caries treatment orprophylaxis. They may also or instead be used for prevention ortreatment of oral malodor. As the microorganisms and fragments thereofaccording to the present invention have a very low, unobtrusive smelland taste, they are particularly suited as sensorically neutral agentsfor preventment of caries, oral biofilm formation including formation ofdental calculus and/or for prevention or treatment of oral malodor. Themicroorganisms and fragments thereof, and also compositions comprisingthen, can thus advantageously be used in food and feed compositions,particularly in pet foods. Furthermore, the invention is concerned withmethods of preparing such microorganisms, fragments, compositions, foodsand feeds.

According to the invention, the microorganism or fragment thereof usefulas a sensorically neutral oral care agent, preferably is a lactic acidbacterium.

As indicated above, it has now surprisingly been found that lactic acidbacteria can be used as sensorically neutral oral care agents,particularly for prophylaxis and treatment of dental calculus, asanti-caries and/or anti-oral malodor agents. This could not be expected,as lactic acid bacteria are commonly known to exhibit a strongly sour oracidic flavor and/or smell, e.g. in yoghurt and other milk products.

According to the present invention, a lactic acid bacterium is anymicroorganism taxonomically of order Lactobacillales, and preferably isof family Lactobacillaceae. A microorganism according to the inventionis considered to belong to a specified taxonomic group if it is moresimilar to a type strain within this taxonomic group or within a lessertaxonomic rank than to any type strain belonging to a taxonomic groupother than the group in question or its lesser ranks, wherein saidanalysis is based on a comparison of their respective genetic materialexcluding plasmids and non-integrated viruses. Thus, a microorganism isconsidered to belong to the family of Lactobacillaceae if it is, asdefined in the previous sentence, genetically more similar to a typestrain of a species or genus within the family of Lactobacillaceaecompared to type strains of a species or genus belonging to anotherfamily within the order of Lactobacillales.

Similarity according to the present invention is assessed using theNeedleman-Wunsch global alignment algorithm. This algorithm is thestandard algorithm to find the optimum alignment including gaps of twonucleic acid sequences along their entire length. The algorithm ofNeedleman and Wunsch has been published in 1970 J. Mol. Biol. 48,443-453, wherein a penalty for a gap of n positions is computedaccording to the formula

gap opening penalty+(n−1)×gap extension penalty.

There is no penalty for hanging ends.

Gap open penalty in the context of the present invention is 10.0. Thegap extension penalty in the context of the present invention is 0.5.The scoring matrix for comparing nucleic acid sequences in the contextof the present invention is the unitary DNA identity matrix, whichassigns a score of 1 for each identical base “substitution”, and −10000for all other base substitutions. Sequence alignments can be performedwith these parameters e.g. via publicly available tools offered by theEBI.

A particular advantage inherent in microorganisms of the family ofLactobacillaceae is that they can generally be considered save forconsumption by human beings and animals. For example, lactic acidbacteria have been used for a long time in the manufacture of foods forexample by processing milk. They are also easy to handle due to theirlow danger to human or animal health and they are easy to cultivate inlarge quantities, e.g. in batches of 500 l of culture medium.Particularly preferred methods of cultivation will be described later.

Among the members of family of Lactobacillaceae, such lactic acidbacteria are particularly preferred according to the present inventionwhich belong to genus Lactobacillus, Paralactobacillus, Pediococcus orSharpea, wherein microorganisms of genus Lactobacillus are mostpreferred. Such microorganisms have been extensively used in thepreparation of foods and feeds; they are easy to handle and can beproduced in large quantities. Furthermore, within the family ofLactobacillaceae it is particularly among genus Lactobacillus thatmicroorganisms can be obtained which are both sensorically neutral andeffective as oral care agents, particularly as anti-dental calculusagents, anti-caries agents and/or anti-oral-malodor agents.

The lactic acid bacteria of the present invention are preferablyrod-shaped or spherical, varying from long and slender to short bentrods, are moreover preferably immotile and/or asporogenous and producelactic acid as a major or sole product of fermentative metabolism. Thegenus Lactobacillus to which the microorganism of the present inventionbelongs in a preferred embodiment is divided up by the followingcharacteristics into three major subgroups, whereby it is envisaged thatthe Lactobacillus species of the present invention can belong to each ofthe three major subgroups:

-   (a) homofermentative lactobacilli    -   (i) producing lactic acid, preferably the L-, D- or DL-isomer(s)        of lactic acid in an amount of at least 85% from glucose via the        Embden-Meyerhof pathway;    -   (ii) growing at a temperature of 45° C., but not at a        temperature of 15° C.;    -   (iii) being long-rod shaped; and    -   (iv) having glycerol teichoic acid in the cell wall;-   (b) homofermentative lactobacilli    -   (i) producing lactic acid, preferably the L- or DL-isomer(s) of        lactic acid via the Embden-Meyerhof pathway;    -   (ii) growing at a temperature of 15° C., showing variable growth        at a temperature of 45° C.;    -   (iii) being short-rod shaped or coryneform; and    -   (iv) having ribitol and/or glycerol teichoic acid in the cell        wall;-   (c) heterofermentative lactobacilli    -   (i) producing lactic acid, preferably the DL-isomer of lactic        acid in an amount of at least 50% from glucose via the        pentose-phosphate pathway;    -   (ii) producing carbondioxide and ethanol    -   (iii) showing variable growth at a temperature of 15° C. or 45°        C.;    -   (iv) being long or short rod shaped; and    -   (v) having glycerol teichoic acid in the cell wall.

Based on the above-described characteristics, the microorganismspreferred according to the present invention can be classified to belongto the group of lactic acid bacteria, particularly to the genus ofLactobacillus.

In a preferred embodiment, the microorganism of the present inventionhas a metabolic fingerprint selected from the group consisting of:

-   (i) it metabolizes D-lactose, but not L-sorbose and/or D-saccharose    and/or D-inuline,-   (ii) it metabolizes inuline,-   (iii) it metabolizes L-sorbose, but not D-lactose and/or    D-saccharose and/or inuline, and-   (iv) it metabolizes L-sorbose, D-lactose and inuline.

Preferably, the microorganism of the present invention has a metabolicfingerprint selected from the group consisting of:

-   (i) it metabolizes D-lactose, but not L-sorbose, D-saccharose and    inuline,-   (ii) it metabolizes L-sorbose, D-lactose and inuline, but not    D-saccharose,-   (iii) it metabolizes L-sorbose, but not D-lactose, D-saccharose and    inuline, and-   (iv) it metabolizes L-sorbose, D-lactose, D-saccharose, but not    inuline.

Of course, the microorganism of the present invention is not limited tothe metabolization of the aforementioned sugars of the metabolicfingerprint patterns, but may be capable of metabolizing further sugars.

Within the present invention, the term “microorganism” not only refersto such microorganisms which, when placed in the appropriate culturingconditions, can multiply (viable microorganisms). It is a particularadvantage of the present invention that instead of such viablemicroorganisms also the corresponding thermally inactivated orlyophilized microorganisms can be used. This is particularly useful assome consumers may object to the idea of consuming viablemicroorganisms, or giving such viable microorganisms to children or petanimals even though they are assured that such viable microorganisms arebeneficial to their health. As the present invention is concerned withincreasing compliance with oral care instructions, it is a particularadvantage that even such unfounded objections can be taken care of.Thus, unless explicitly mentioned this description and the accompanyingexamples, figures and claims do not differentiate between viable,thermally inactivated or lyophilized microorganisms.

According to the present invention, thermally inactivated cellspreferably are obtained by autoclaving viable microorganism cells at atemperature of 121° C. for at least 20 minutes in the presence ofsaturated steam at an atmospheric pressure of 2 bar. Such preparation ofthermally inactivated microorganisms can be achieved using standardlaboratory equipment, as the process of autoclaving is a standardtechnique known in the art of microbiology and correspondingbiotechnological engineering. It is a very fast technique and is provento inactivate microorganisms of family Lactobacillaceae and particularlyof genus Lactobacillus.

Alternatively, thermal inactivation of microorganisms according to thepresent invention can be achieved by freezing such cells to atemperature of −20° C. Such freezing is also easy to perform usingstandard laboratory equipment.

Regardless of the mode of thermal inactivation, i.e. by autoclaving orby freezing, it is according to the present invention preferred that theconcentration of viable microorganisms is reduced by the treatment by atleast 85%, 90% or 95%, and particularly preferred by at least 97%, 98%,99% and more particularly preferred 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8% or 99.9%. Particularly preferred is suchautoclavation or freezing that reduces the concentration of viablemicroorganisms by at least 3 orders of magnitude (i.e. by a factor of1000), even more preferably by at least 4 orders of magnitude and evenmore preferably by at least 5 orders of magnitude.

A particular advantage of the microorganisms of the present invention isthat they retain their usefulness as sensorically neutral oral careagents, preferably anti-dental calculus agent, anti-caries and/oranti-oral-malodor agents even after such inactivation by autoclaving orfreezing. In such thermally inactivated form, the microorganisms of thepresent invention are particularly easy to store even at 20° C. in astandard atmosphere having 80% relative humidity at this temperature.This storability is of particular importance, as the inactivatedmicroorganisms according to the present invention allow to produce foodsor feeds comprising such inactivated forms without corrupting the “bestbefore” date.

The same advantages can be obtained according to the present inventionby using lyophilized microorganisms of the present invention, i.e.lyophilized microorganisms of family Lactobacillaceae and particularlylyophilized lactic acid microorganisms of genus Lactobacillus.Lyophilization according to the present invention is preferably for atleast 2 hours at room temperature, i.e. at a temperature between 16° C.and 25° C.

In addition to or alternatively to microorganisms of the presentinvention, for example in viable, thermally inactivated or lyophilizedform, fragments thereof can be used. A fragmentation of microorganismscan be easily performed using standard methods known in the art,particularly by cell lysis and/or pasteurization. The method used forlysing or fragmenting a microorganism according to the present inventionincluding thermally inactivated and/or lyophilized forms thereof is ofno particular concern. Appropriate methods can be chosen for exampleamong chemical methods including enzymatic treatment, preferably by oneor more proteases, for example by proteinase K, lipases or glycosidases;non-limiting examples for other chemicals are ionophores, detergents,for example sodium dodecyl sulfate, acids or bases; non-limitingexamples of physical means are high pressure, like French-pressing,osmolarity and milling for example using glass or iron beats.Particularly preferred methods for producing thermally inactivatedand/or lyophilized microorganisms according to the present invention andfragments thereof are described in WO 2006/027265 A1, pages 25-28 and WO2009/149816 A1, pages 28-34. “A fragment of the microorganism of thepresent invention” encompasses any part of the cells of themicroorganism of the present invention. Preferably, said fragment is amembrane fraction obtained by a membrane-preparation. Membranepreparations of binder microorganisms belonging to the family ofLactobacillaceae, preferably to the genus of Lactobacillus, can beobtained by methods known in the art, for example, by employing themethod described in Rollan et al. Int. J. Food Microbiol. 70 (2001),303-307, Matsuquchi et al, Clin. Diagn. Lab. Immunol. 10 (2003), 259-266or Stentz et al. Appl. Environ. Microbiol. 66 (2000), 4272-4278 orVarmanen et al. J. Bacteriology 182 (2000), 146-154. Alternatively, awhole cell preparation is also envisaged. Preferably, the hereindescribed fragment of the binder microorganism of the present inventionretains the capability of specifically binding to a mutans Streptococcusand more preferably Streptococcus mutans, which is described in detailherein.

The microorganisms used in the present invention, i.e. lactic acidbacteria preferably of family Lactobacillaceae or of genus Lactobacillusincluding respective thermally inactivated or lyophilized forms thereofand fragments thereof, are preferably used as anti-caries agents.According to the present invention, the term “anti-caries agent” isdefined as any agent which, by its mere presence in the oral cavity of ahuman or animal including pet animals like dogs, cats, rats, mice,hamsters, guinea pigs or monkeys, reduces the risk of developing newcariotic lesions. It is a particular advantage of the present inventionthat the microorganism used as anti-caries agent does not have to beantimicrobially effective on its own. Thus, it is not necessaryaccording to the present invention that microorganisms or fragmentsthereof used as anti-caries agents reduce in a co-cultivation the numberof caries-generating microorganisms, particularly microorganismsbelonging to the group of mutans Streptococci.

It is therefore another advantage according to the present inventionthat the microorganisms used as anti-caries agents have a very limitedimpact on the normal microflora of the oral cavity of a human being orof an animal, particularly a pet animal. Suitable microorganisms will bedescribed below.

The microorganism used in the present invention, i.e. lactic acidbacteria preferably of family Lactobacillaceae or of Genuslactobacillus, including respective thermally inactivated or lyophilizedforms thereof and fragments thereof, are preferably used as anti-dentalcalculus agents. According to the present invention, the term“anti-dental calculus agent” is defined as any agent which, by its merepresence in the oral cavity of a human or animal including pet animals,reduces the risk of developing dental calculus, or which slows downfurther development of dental calculus. It is not required that theanti-dental calculus agent of the present invention actually removesdental calculus already formed on a tooth.

Also, the microorganisms used in the present invention, i.e. lactic acidbacteria preferably of family Lactobacillaceae or of Genus lactobacillusincluding respective thermally inactivated or lyophilized forms thereofand fragments thereof, are preferably used as anti-oral malodor agents.According to the present invention, the term “oral malodor” indicatesany unpleasant smell originating in the oral cavity. The term thus isused according to the present invention both for weak forms of malodor,also termed “bad breath”, which are not considered an illness, and alsofor such forms of malodor which, for example due to their intensity orscent, are considered to be the symptom of an illness; such latter formsof oral malodor are also called halitosis. Clinically relevant forms oforal malodor, i.e. halitosis, can have various causes. Most notable forall forms of oral malodor are infections by microorganisms colonizingthe oral cavity, wherein such microorganisms digest organic material,for example dead epithelial cells, other microorganisms or residual foodor feed. Preferably, the present invention is set to delay onset of oralmalodor, and/or to reduce the intensity or alter the scent of oralmalodor, be it clinically not relevant bad breath or clinically relevanthalitosis. However, unless explicitly mentioned herein, the term “oralmalodor” does not cover malodors originating beyond the oral cavity,e.g. in the esophagus or stomach. Such malodors may for example becomenoticeable during eructation.

Within the present invention, the term “oral cavity” indicates thatcavity which extends from lips and teeth up to but not including theuvula.

The mouth defines the oral cavity of mammals, preferably humans oranimals such as pets, composed by the oral mucosa (gums, lips, cheeks,palate and floor of the mouth), the tongue and the teeth (includingartificial structures).

Also according to the present invention, the term “sensorically neutral”is defined as such agent which can replace up to 2 wt.-% of wholemealwheat flour such that at most 5 out of 10 trained panelists willconsider the taste and scent of such mixture of wholemeal wheat flourand microorganisms and/or fragments according to the invention to benoticeably altered, judging on the basis of a scale ranging from nodifference, slightly altered, noticeably altered to clearly altered.Preferably, up to 5 wt.-% of wholemeal wheat flour can be replaced bythe microorganisms and/or fragments thereof such that at most 5 out of10 panelists consider the taste and/or scent of such mixture to benoticeably altered.

According to the invention, there is thus also provided a compositioncomprising

-   -   a binder microorganism or fragment thereof in an amount both (a)        sensorically neutral and (b) effective for reducing mutans        Streptococci in the oral cavity of a human or animal and/or        effective for reducing oral malodor, wherein the microorganism        is a lactic acid bacterium.

It has now surprisingly been found that to exert an anti-caries effectby a sensorically neutral agent, it is sufficient to use a binder lacticacid bacterium for reducing mutans Streptococci in the oral cavity. Asdescribed above, such binder lactic acid bacteria, thermally inactivatedor lyophilized forms thereof and also fragments of such binder lacticacid bacteria can be used as anti-caries agent without having ananti-microbial effect, i.e. without killing cariogenic microorganismsthemselves or repressing their growth. Instead, it is sufficient that abinder microorganism or fragment thereof binds to one or more species ofmutans Streptococci. Such binding can lead to an agglutination of boundmutans Streptococci, which in turn are removed from the oral cavity bynormal swallowing of saliva and during eating and drinking. As describedin WO 2008/074473 A2 removal of mutans Streptococci from the oral cavitysignificantly reduces the risk of biofilm formation in the oral cavityand on teeth, thereby indirectly reducing the risk of developing caries.This particularly holds true also for such binder microorganisms whichbind to Streptococcus mutans, as described in WO 2006/027265 A1.However, it had not been known so far that such binder microorganismscould be used as sensorically neutral agents, i.e. in a sensoricallyneutral concentration as described above and detailed hereinafter. Also,it had not been known so far that such binding to mutans Streptococciand particularly to Streptococcus mutans could reduce the risk ofdevelopment of oral malodor.

According to the invention, a composition is thus preferred comprising amicroorganism or fragment thereof that is capable of specificallybinding to a bacterium belonging to the group of mutans Streptococci,wherein the specific binding is

-   (i) resistant to heat treatment; and/or-   (ii) resistant to protease treatment; and/or-   (iii) calcium-dependent; and/or-   (iv) formed within a pH range between 4.5 and 8.5; and/or-   (v) formed in the presence of saliva.

The term “specifically binding” in the context of the present inventionmeans that the binder microorganism or fragment thereof, preferably amicroorganism (or corresponding fragment) belonging to the family ofLactobacillaceae and more preferably of genus Lactobacillus, binds toone or more strains belonging to mutans Streptococci, preferably toStreptococcus mutans, but does not bind to most other, preferably to noother species belonging to the genus Streptococcus. Namely, the bindermicroorganism or fragment thereof does preferably not bind to bacteriabelonging to the species of Streptococcus oralis and/or Streptococcusmitis and/or Streptococcus sanguinis. Even more preferably, the bindermicroorganism also does not bind to bacteria belonging to the species ofStreptococcus salivarius, more preferably belonging to the subspeciesthermophilus. More preferably, the binder microorganism or fragmentthereof does not bind to Streptococcus oralis DSMZ 20066, Streptococcusoralis DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitisDSMZ 12643 and/or Streptococcus sanguinis DSMZ 20567. And even morepreferably, the binder microorganism or fragment thereof also does notbind to Streptococcus salivarius ssp. thermophilus,

The specific binding reaction comprises binding and, preferably,aggregating Streptococcus mutans or other mutans Streptococci cells asdescribed herein by the binder microorganism of the present inventionincluding a fragment thereof in the mouth. This specific binding leads,in consequence, to flushing away the boud cells by, for example,salivary flow or by a mouth rinse or mouth wash and the like asdescribed herein. Preferably, the specific binding reaction of thebinder microorganisms of the present invention and their fragments toStreptococcus mutans and/or other mutans Streptococci prevents suchStreptococcus cells from attaching to the surface of a tooth or teeth,or, while not being bound by such theory, could lead to detachment ofStreptococcus cells from the surface of a tooth or teeth. Inconsequence, the specific binding reaction results in flushing awaybound Streptococcus cells out of the mouth, thereby diminishing acausative agent of biofilm formation and, thus, preventing and/ortreating caries.

It is believed that the binder microorganism or fragment thereof maybind specifically to the streptococcal antigen I/II which is also knownas antigen B, IF, P1, SR, MSL-1 or PAc. However, the bindermicroorganism or fragment thereof may bind to any other protein orsurface structure of S. mutans, thereby aggregating S. mutans andflushing it out of the oral cavity as described herein. It is known thatStreptococcus mutants binds via said streptococcal antigen I/II to thepellicle. Accordingly, when the binder microorganism of the presentinvention may bind, for example, to said streptococcal antigen I/II,Streptococcus mutans or another respective mutans Streptococcus ishampered to bind to the surface of teeth which thus helps to preventand/or treat caries.

The pellicle is a clear, thin covering containing proteins and lipidsfound in saliva. It is formed within seconds after a tooth surface iscleaned. Pellicle formation is the first step in dental plaqueformation. Dental plaque is a soft deposit that accumulates on theteeth. Plaque can be defined as a complex microbial community, withgreater than 10¹⁰ bacteria per milligram. It has been estimated that asmany as 400 distinct bacterial species may be found in plaque. Inaddition to the bacterial cells, plaque contains a small number ofepithelial cells, leukocytes, and macrophages. The cells are containedwithin an extracellular matrix, which is formed from bacterial productsand saliva. The extracellular matrix contains protein, polysaccharideand lipids. One of the proteins present in saliva is agglutinin which ison the one hand thought to lead to a partial removal of mutansStreptococci from the mouth, however, is on the other hand suspected tofacilitate adhesion of mutans Streptococci to the surface of teeth,thereby facilitating the initial attachment of Streptococcus cells toteeth and, thus, onset of caries.

Preferably, the above mentioned binder microorganism belonging to thegroup of lactic acid bacteria—or fragment thereof—is capable ofspecifically binding to Streptococcus mutans serotype c (DSMZ 20523)and/or serotype e (NCTC 10923) and/or serotype f (NCTC 11060) and/orStreptococcus sobrinus DSMZ 20742 and/or Streptococcus ratti DSMZ 20564and/or Streptococcus cricetus DSMZ 20562 and/or Streptococcus ferus DSMZ20646 and/or Streptococcus macacae DSMZ 20714.

This means that the above mentioned binder microorganism belonging tothe group of lactic acid bacteria, or its fragment, preferably binds toat least one microorganism selected from the group consisting ofStreptococcus mutans serotype c (DSMZ 20523), serotype e (NCTC 10923),serotype f (NCTC 11060), Streptococcus sobrinus DSMZ 20742,Streptococcus ratti DSMZ 20564, Streptococcus cricetus DSMZ 20562,Streptococcus ferus DSMZ 20646 and Streptococcus macacae DSMZ 20714.More preferably, the above mentioned binder microorganism belonging tothe group of lactic acid bacteria or fragment thereof binds to anycombination, grouping or subgrouping of the above mentioned bacteria.Even more preferably, the above mentioned binder microorganism orfragment thereof belonging to the group of lactic acid bacteria binds toall of the above mentioned bacteria. In accordance with the presentinvention a “serotype” is an antigenic property of a bacterial cell,preferably of a Streptococcus mutans or Streptococcus sobrinus cell,identified by serological methods known in the art.

As described above, the specific binding of the binder microorganism orfragment thereof to mutans Streptococci and preferably to Streptococcusmutans is preferably resistant to heat treatment. Accordingly, bindingis not abolished when the binder microorganism of the present inventionis treated with heat, for example, at a temperature above 55° C., evenmore preferably of more than 65° C., particularly preferred of more than95° C. and most preferred at 121° C. After cooling down, the capabilityof the binder microorganism of the present invention or its fragment tospecifically bind mutans Streptococci is determined as described herein.

The corresponding temperature can depend on the specific bindermicroorganism species but can be easily determined by the skilled personby routine experimentation, e.g. by incubating the corresponding cellsat different temperatures and determining the amount of binder cells orfragments thereof which is still capable of specifically binding tomutans Streptococci and/or Streptococcus mutans by using methods asthose shown herein. Generally, the heat treatment should last for aperiod of time of at least 1 minute. Preferably, the heat treatmentlasts for a period of time of at least n minutes, wherein n is aninteger in the range of 2 to 60, with n=20 being particularly preferred.However, there is in principle no upper limit for the time ofincubation. However, it is preferably no longer than 4, 3, 2 or 1hour(s). The most preferred heat treatment is at least 20 minutes at atemperature of 121° C. in a saturated steam having an atmosphericpressure of 2 bar. Thus, the thermally inactivated form of the bindermicroorganism of the present invention obtainable by autoclaving isparticularly preferred. The most preferred heat treatment is consideredas abolishing any function of a protein and of any vitality of cellswhich thus distinguishes the microorganism of the present invention fromother microorganism in that it is still capable of the specificallybinding to S. mutans. Hence, it is very useful for any food, feed, drinkor composition of the present invention if it is desired that themicroorganism should not be alive.

The specific binding of the binder microorganism or its fragment isfurthermore preferably characterized by its resistance to proteasetreatment. Preferably, the binding is resistant to treatment with one ormore proteases selected from the group consisting of pronase E,proteinase K, trypsin and chymotrypsin. These proteases show nospecificity and, thus, are considered as degrading any protein being onthe cell surface of a microorganism. Other proteases, which are known tohave preferences for certain patterns of amino acid residues, areelastase elastase, thrombin, aminopeptidase I, carboxypeptidase,dostripain, endoproteinase, papain, cathepsin B, pepsin, gastricsin,chymosin, cathepsin D. The latter proteases could also be used to testwhether the specific binding of the binder microorganism or fragmentthereof to S. mutans or another mutans Streptococcus is resistant to thelatter more specific proteases. Thus, after protease treatment which isdescribed in the examples of WO 2008/074473 A2 or WO 2006/027265 A1,which are incorporated herein, the binder microorganism or fragment isstill capable of specifically binding to Streptococcus mutans/mutansStreptococci.

In addition, the specific binding of the binder microorganism orfragment is furthermore preferably characterized by its dependency oncalcium. Preferably, the specific binding takes place in the presence ofa concentration of calcium ions between 0.05 mM and 500 mM, preferablybetween 1 mM and 100 mM. Particularly preferred the calciumconcentration is between 2 mM and 30 mM. The dependency of the specificbinding on calcium can be tested as described in the examples of WO2008/074473 A2 or WO 2006/027265 A1, which are incorporated herein.Moreover, the specific binding to the binder microorganism or fragmentis preferably maintained over a pH range between 4.0 and 9.0, preferablybetween 4.0 and 7.0 In particular, the pH value at which the specificbinding takes still place is preferably 4.5. Assaying of the maintenanceof the specific binding over the pH range described above is shown inthe above mentioned examples.

The specific binding is preferably independent of magnesium. Thus, it isnot necessary that magnesium ions or magnesium salts are present.

Another preferred characteristic of the specific binding is itsoccurrence in the presence of saliva. Saliva is an exogenous secretewhich is synthesized by the salivary glands. It is a complex liquidcontaining, apart from about 99% water a multiplicity of organic andinorganic compounds. Physiological ingredients of saliva are, interalia, enzymes, e.g., amylases, carboanhydrases, lysozyme, peroxidases orproteins, e.g., mucins, lactoferrin, proline-rich proteins, cystatines,histatines or statherines or soluble IgA. Thus, although a variety ofpotentially interfering substances are present in saliva, the specificbinding of the microorganism of the present invention was not disturbedor hampered. For testing the specific binding in the presence of saliva,it is preferred that saliva is used which contains preferably theStreptococcus species described above. If, however, Lactobacillusrhamnosus species are tested for specific binding to S. mutans in thepresence of saliva, it is preferred that Streptococcus salivarius ssp.thermophilus is omitted. The specific binding is assayed as describedherein. The aforementioned characteristics of the binder microorganismor fragment thereof belonging to the group of lactic acid bacteriarenders it to be a robust and effective agent for preventing and/ortreating caries since it is mainly administered in various forms to themouth including the oral cavity and teeth where, inter alia, salivaincluding certain proteases and low pH values after ingestion ofcarbohydrate containing food stuff is present. Moreover, the resistanceto heat has beneficial effects in adding the microorganism of thepresent invention as additive to food or feed during the preparation ofsaid food or feed. Namely, food or feed is often heat sterilized,pre-cooked, pasteurized and the like which is detrimental for viabilityof microorganisms.

The binder microorganism of the present invention, and/or thecorresponding fragment thereof, is capable of specifically binding toone or more mutans Streptococci and preferably to Streptococcus mutans,wherein the specific binding is

-   (i) resistant to heat treatment, and-   (ii) resistant to protease treatment, and-   (v) formed in the presence of saliva.

Such binder microorganisms and fragments thereof combine theaforementioned advantages. In particular, since their specific bindingis resistant to heat treatment, they can be incorporated in a productthat is heat treated for sterilization, thereby prolonging shelf life ofthe product without corrupting the oral health properties conferred tothe product by the microorganisms of fragment thereof, preferablywithout corrupting the ant-dental calculus effectiveness, theanti-caries effectiveness or the ant-oral malodor effectiveness of theproduct. Also, due to the protease resistance the binder microorganismsand fragments can be incorporated in various products of differentcomposition even before such products are heat treated for proteindenaturation. And importantly the formation of a specific binding in thepresence of saliva allows to use the binder microorganisms and fragmentsthereof also in such products which do not drain saliva from the oralcavity. This ability in turn increases consumer compliance, particularlyin children and animals including pet animals, as such consumers tend tobe reluctant to use a product which would lead to a dry oral cavity.

For these reasons, the binder microorganism of the present invention,and/or the corresponding fragment thereof, is even more preferablycapable of specifically binding to one or more mutans Streptococci andpreferably to Streptococcus mutans, wherein the specific binding is

-   (i) resistant to heat treatment for 20 minutes at a temperature of    121° C. in a saturated steam having an atmospheric pressure of 2    bar, and-   (ii) resistant to treatment by one or more enzymes selected from    pronase E, proteinase K, trypsin and chymotrypsin, and-   (v) formed in the presence of saliva.

Preferably, the specific binding of the binder microorganism of thepresent invention, and/or the corresponding fragment thereof is also

-   (iii) calcium-dependent, and-   (iv) formed within a pH range between 4.5 and 8.5.

The method to determine the binding of binder microorganisms andfragments thereof to mutans Streptococci is described in Lang et al(2010) Journal of Dental Research 89(2) 175-179, which is incorporatedherein.

Preferably, the specific binding of the binder microorganism or fragmentthereof can be assayed as follows:

-   (a) growing said binder microorganism to stationary phase, or, in    case a fragment is to be tested, obtaining such fragment,-   (b) mixing said binder microorganism or fragment with a mutans    Streptococcus which has been grown to stationary phase,-   (c) incubating the mixture obtained in step (b) under conditions    allowing the formation of aggregates of said microorganism and said    Streptococcus, and-   (d) detecting aggregates by the occurrence of a pellet.

In a preferred embodiment the bacterium belonging to the group of mutansStreptococci used in such an assay is Streptococcus mutans. For aspecific binding to mutans Streptococci, preferably no binding can bedetected to at least one, preferably at least two and more preferably atleast three and even more preferably all microorganisms selected fromthe group consisting of Streptococcus salivarius ssp. thermophilus,Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395,Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 andStreptococcus sanguinis DSMZ 20567.

In particular, binder microorganisms belonging to the group of lacticacid bacteria, preferably of family Lactobacillaceae and even morepreferably of genus Lactobacillus, or corresponding fragments, arepreferably mixed with mutans Streptococci in cell-to-cell ratios of 3:1to 60:1 (mutans Streptococci:binder microorganism). Both the lactic acidbinder bacteria and mutans Streptococci are grown in liquid culture tostationary phase. Preferably, the optical density is measuredphotometrically at a wavelength of 600 nm.

The mentioned ratios correspond to a ratio of colony forming units from1:50 to 1:2.5. Preferably, an OD600=1 in 1 ml corresponds to 3×10⁸colony forming units of a mutans Streptococcus. Preferably, an OD600=1in 1 ml corresponds to 7×10⁹ colony forming units of lactic acidbacteria. Preferably, for assaying the aggregation reaction bypelleting, the bacteria are in a volume of 2 ml in 15 ml Falcon tubes.If necessary, the culture suspensions are diluted with PBS-butter toobtain volumetric ratios mentioned above, while keeping the final volumeat 2 ml.

Preferably, the mixture of Streptococcus and binder lactic acid bacteriaor fragments thereof is vortexed for about 15 seconds and then leftundisturbed for at least 5, 10, 15 minutes and more preferably for 20minutes at room temperature, i.e. any temperature between 16° C. and 25°C. An aggregation is visible as an immediate turbity of the suspensionand, after at least 20 minutes, an aggregation is visible by aggregatesthat settle as a visible pellet, whereas non-mutans Streptococcusaggregating mixtures stay in suspension. As a control, self-aggregationof the respective binder bacterium or fragment and the mutansStreptococcus strain can be assayed by omitting either the mutansStreptococcus or the binder microorganisms/fragment.

The aggregation of a binder microorganism and a mutans Streptococcusaccording to the above described assay can be quantified by separatingthe formed aggregates as described by Lang et al (2010) Journal ofDental Research 89(2) 175-179, or by centrifugation, e.g. at 500×g for30 seconds. Subsequently, the amount of aggregation can be determined bymeasuring the amount of non-aggregated cells that are left in thesupernatant. The determination can be carried out by any suitable meansknown to the person skilled in the art. Preferably, the determination iscarried out by removing a certain volume of the supernatant, e.g. 1 ml.Subsequently, the optical density of the removed supernatant may bemeasured at any suitable wavelength, known to the skilled person, e.g.at 600 nm. The measured value after subtraction of a value acorresponding control test without lactobacilli represents the amount ofcells that have not been aggregated.

A method for determining specific binding according to the presentinvention is described in example 4 of WO 2008/74473 A2, withcorresponding instructions for cultivation of respective microorganismsgiven in example 1 of that publication. Both examples are incorporatedherein for the purpose of disclosing a method for determining specificbinding according to the present invention.

Alternatively, in order to address the possible problem ofself-aggregation a stain, preferably a fluorescent stain, can beemployed. Thus, in a more preferred embodiment, the specific binding canbe assayed as follows:

-   (a) growing said microorganism to stationary phase;-   (b) mixing said microorganism with a bacterium belonging to the    group of mutans Streptococci which has been grown to stationary    phase and which has been stained using a suitable stain, preferably    a fluorescent stain;-   (c) incubating the mixture obtained in step (b) under conditions    allowing the formation of aggregates of said microorganism and a    bacterium of the group of mutans Streptococci; and-   (d) detecting aggregates by the detection of the stain, preferably a    fluorescencent stain.

Again, in a preferred embodiment the bacterium belonging to the group ofmutans Streptococci used in such an assay is Streptococcus mutans.

Preferably the aggregation assay may be carried out in that first thebinder microorganism and mutans Streptococci are grown to stationaryphase as described above. Preferably, the optical density is measuredphotometrically at a wavelength of 600 nm. Preferably, an OD600=1 in 1ml corresponds to 3×10⁸ colony forming units of a respective mutansStreptococcus. Preferably, an OD600=1 in 1 ml corresponds to 7×10⁹colony forming units of binder microorganism, particularly of familylactobacillaceae and even more preferably of genus lactobacillus, asdescribed herein.

Subsequently, the mutans Streptococci are stained. In a furtherpreferred embodiment, the binder microorganisms are stained, whereas theStreptococci are not stained. As stain any suitable stain can be used,preferably a fluorescence stain known to the person skilled in the artmay be used. Preferably, a specific or unspecific fluorescence stain maybe used, for example, CFDA-SE to stain intact cells, other useful stainsinclude carboxyfluorescein diacetate acetoxymethyl ester, BCECF AM andCalcein AM. Specifically, the cells are harvested, e.g. bycentrifugation, preferably at 3200×g for 5 min. Subsequently, theobtained pellet may be resuspended in any suitable buffer known theperson skilled in the art, preferably in a PBS-buffer. The amount ofbuffer may be calculated so that the resulting suspension has an OD600of, e.g., 4.2/ml. Subsequently, the suspension may be mixed with asuitable stain, e.g. a fluorescence stain, preferably with5,6-carboxyfluorescein diacetate, succhinimidyl ester (CFDA-SE), morepreferably with 2 μl of a CFDA-SE solution (Invitrogen). Subsequently,the cells may be incubated for a suitable time period, as known to theskilled person, e.g. for 2 hours, at a suitable temperature as known tothe skilled person, for instance, at 37° C. In a further step, thestained cells may be harvested, e.g. by centrifugation. Preferably, thecentrifugation is carried out at 3200×g for 5 min. The cells may then beresuspended in a suitable buffer, as known to the person skilled in theart, e.g. in 2 ml of a PBS-buffer.

For the aggregation, binder microorganisms preferably mixed with mutansStreptococci in volumetric ratios of 3:1 to 1:3 (mutansStreptococci:binder microorganism). More preferably, the volumetricratio of the mixture is 1:1. The mentioned ratios correspond to a ratioof colony forming units from 1:50 to 1:150. For assaying the aggregationreaction via measuring of staining, preferably of fluorescence, thebinder microorganism and the mutans Streptococci are used in anysuitable volume known to the skilled person, preferably, in a volume of50 μl. Preferably, the mixture is carried out in a microtiter plate,e.g. in a 96 well microtiter plate. Subsequently, the mixture may bevortexed, preferably for 12 min at full speed. Afterwards, the mixturemay be centrifuged, e.g. for 10 seconds at 500×g. The supernatant maythen be removed and the pellet may be resuspended in any suitable bufferknown the person skilled in the art, preferably in PBS-buffer in anysuitable volume, e.g. in 100 μl. The staining of the suspension may bemeasured in the mixture by any suitable means known to the skilledperson. Preferably, in case of fluorescence, the fluorescence may bedetected in a fluorescence reader, e.g. at a wavelength of 495 nm forexcitation and 525 nm for emission. As controls, binder microorganismalone and stained mutans Streptococci alone may be assayed. Anybackground staining, e.g. fluorescence, may be measured for the testedmutans Streptococci alone and may preferably be subtracted from thevalue for the aggregation with the respective binder microorganism. Anaggregation effect is present if the background staining, e.g.fluorescence, measured as indicated herein above, is subtracted from themeasured staining, e.g. fluorescence, in a sample containing a bindermicroorganism as described herein above and a tested mutansStreptococcus, as described herein above, and the resulting value is atleast above zero. More preferably, an aggregation effect is present ifthe resulting value is reproducibly above zero in a series of tests,carried out as described herein above. A “series of experiments” meansat least 2, preferably 3, more preferably 4 and most preferably 5 tests.

The alternative method of testing binding can be performed as describedin example 5 of WO 2008/074473 A2, which is incorporated herein byreference.

For fragments of binder microorganisms, binding is essentiallydetermined as for viable or inactivated binder microorganisms asdescribed above. The amount of fragments to be used for determiningbinding or for preparing a food or feed composition according to theinvention instead of binder microorganism cells is preferably the sameas the amount of cell wall material of viable or thermally inactivatedbinder microorganism. For example, peptidoglycan content of bindermicroorganism content can be measured by using appropriate dyes, and thesame amount of fragments based on such dye measurement can be used.

The above mentioned binder microorganisms are preferably lactic acidbacteria belonging to the genus of Lactobacillus, more preferablyLactobacillus species as described herein. Even more preferably saidLactobacillus belongs to the species of Lactobacillus paracasei orLactobacillus rhamnosus. However, the Lactobacillus species are notlimited thereto. The above mentioned binder microorganisms maypreferably be “isolated” or “purified”. The term “isolated” means thatthe material is removed from its original environment, e.g. the naturalenvironment if it is naturally occurring. For example, anaturally-occurring microorganism, preferably a Lactobacillus species,separated from some or all of the coexisting materials in the naturalsystem, is isolated. Such a microorganism could be part of acomposition, and is to be regarded as still being isolated because inthat composition is not part of its natural environment. Thus, amicroorganism grown in a pure culture is still considered an isolatedmicroorganism. Likewise, a fragment of a microorganism is considered“isolated” according to the invention if it is separated from at leastsome material of the respective microorganism.

The term “purified” does not require absolute purity; rather, it isintended as a relative definition. Individual microorganisms obtainedfrom a library have been conventionally purified to microbiologicalhomogeneity, i.e. they grow as single colonies when streaked out on agarplates by methods known in the art. Preferably, the agar plates that areused for this purpose are selective for lactic acid bacteria, andparticularly preferably to Lactobacillus species. Such selective agarplates are known in the art.

To obtain fragments, it is preferred to rupture viable or thermallyinactivated binder microorganism cells by methods known in the art, forexample sonication, French press or ball milling, and to separate thefragments from other cell remains by centrifugation. The fragment pelletcan then be washed and centrifuged again to obtain a fragments pellet.

More preferably, the above mentioned binder microorganism belonging tothe group of lactic acid bacteria is selected from the group consistingof Lactobacillus paracasei or Lactobacillus rhamnosus, respectively,having DSMZ accession number DSMZ 16667 (L. paracasei ssp. paracaseiLb-Ob-KI), DSMZ accession number DSMZ 16668 (L. paracasei ssp. paracaseiLb-Ob-K2), DSMZ accession number DSMZ 16669 (L. paracasei ssp. paracaseiLb-Ob-K3), DSMZ accession number DSMZ 16670 (L. paracasei ssp. paracaseiLb-Ob-K4), DSMZ accession number DSMZ 16671 (L. paracasei ssp. paracaseiLbOb-K5), DSMZ accession number DSMZ 16672 (L. rhamnosus Lb-Ob-K6) andDSMZ accession number DSMZ 16673 (L. rhamnosus Lb-Ob-K7) or a mutant orderivative thereof, wherein said mutant or derivative retains thecapability to specifically bind to mutans Streptococci. The term“Lactobacillus paracasei or Lactobacillus rhamnosus having DSMZaccession number” relates to cells of a microorganism belonging to thespecies Lactobacillus paracasei or Latobacillus rhamnosus deposited withthe Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH (“DSMZ”)on Aug. 26, 2004 and having the following deposit numbers DSMZ 16667,16668, 16669, 16670, 16671, 16672 or 16673. The DSMZ is located at theMascheroder Weg 1b, D-38124 Braunschweig, Germany. The aforementionedDSMZ deposits were made pursuant to the terms of the Budapest treaty onthe international recognition of the deposit of microorganisms forpurposes of patent procedure.

“A mutant or derivative” of the above mentioned binder microorganismbelonging to the group of lactic acid bacteria, preferably of thedeposited Lactobacillus paracasei or Lactobacillus rhamnosus cells, haspreferably the same characteristics as the respective deposited strains,i.e. it retains the capability to specifically bind to mutansStreptococci, preferably with the binding characteristics as describedherein. For example, said derivative can be genetically engineered. Inthe context of the present invention the term “genetically engineered”is used in its broadest sense for methods known to the person skilled inthe art to modify desired nucleic acids in vitro and in vivo such thatgenetic modifications are affected and genes are altered by recombinantDNA technology. Accordingly, it is preferred that said methods comprisecloning, sequencing and transformation of recombinant nucleic acids. Forthis purpose appropriate vectors including expression vectors forLactobacillus species as, for example, described in EP 0 506 789 B1, EP0 316 677 B1, EP 0 251 064 B1, EP 0 218 230 B1, EP 0 133 046 B1 or WO89/01970.

Primers, enzymes, further host cells for cloning of intermediateconstructs and the like can be used and are known by the skilledartisan. Preferably, genetically engineered mutants comprise cells of abinder microorganism belonging to the group of lactic acid bacteria,preferably of the family of lactobacteriaceae, even more preferably ofgenus lactobacillus and most preferably one of the depositedLactobacillus species, harbouring a recombinant nucleic acid eithercomprised in their bacterial chromosome or on one or more plasmids orcomprised in their bacterial chromosome and/or one or more plasmids.Said recombinant nucleic acids are preferably foreign to the abovementioned binder microorganism belonging to the group of lactic acidbacteria. By “foreign” it is meant that the polynucleotide or nucleicacid molecule is either heterologous with respect to the host cell, thismeans derived from a cell or organism with a different genomicbackground, or is homologous with respect to the host cell but locatedin a different genomic environment than the naturally occurringcounterpart of said nucleic acid molecule. In this case the heterologouspolynucleotide may be either under the control of its own promoter orunder the control of a heterologous promoter. The above described vectoror nucleic acid molecule, which is present in the host cell may eitherbe integrated into the genome of the host cell or it may be maintainedin some form extrachromosomally. In this respect, it is also to beunderstood that the above described nucleic acid molecule can be used torestore or create a mutant gene via homologous recombination. Plasmidsmay be low, medium or high copy number plasmids. Said geneticallyengineered mutants may harbour nucleic acids encoding a glucanase ormutanase which is capable of degrading the mutan specific 1,3-glycosidicbond of saccharose subunits. Fungal glucanases are, for example,described in Fuglsang et al., J. Biol. Chem. 275 (2000), 2009-2018. Itis also envisaged that genetically engineered mutants comprise cellsharbouring recombinant nucleic acids encoding antibodies which arepreferably secreted or anchored in the bacterial cell wall. The term“antibody” encompasses intact antibodies as well as antibody fragmentsthereof, like, separated light and heavy chains, Fab, Fab/c, Fv, Fab′,F(ab′)2. The term “antibody” also comprises humanized antibodies,bifunctional antibodies and antibody constructs, like single chain Fvs(scFv) or antibody-fusion proteins. It is also envisaged in context ofthis invention that the term “antibody” comprises antibody constructswhich may be expressed in cells of the derivative of the above mentioneddeposited microorganism, e.g. antibody constructs which may betransformed via, inter alia, vectors by methods known in the art. It isin particular envisaged that such antibody constructs specificallyrecognize, for example, the streptococcal antigen I/II. Such an approachis, for example, described in Krueger et al., Nat. Biotechnol. 20(2002), 702-706 or Shiroza, Biochim Biophys Acta 1626 (2003), 57-64.

Secretion of the expressed antibody is preferably achieved byoperatively linking the nucleic acid encoding an antibody to a secretionsignal sequence. Anchoring in the bacterial cell wall could be achievedby making use of the mechanism of the enzyme sortase. Namely, surfaceproteins of gram-positive bacteria are linked to the bacterial cell wallby a mechanism that involves cleavage of a conserved Leu-Pro-X-Thr-Gly(LPXTG) motif and that occurs during assembly of the peptidoglycan cellwall. Accordingly, the nucleic acid molecule encoding an antibody may befused to a sequence encoding the aforementioned conserved motif, whichis used by sortase to anchor proteins in the bacterial cell wall.

It is also envisaged that the above mentioned binder microorganismbelonging to the group of lactic acid bacteria be genetically modifiedto harbor a nucleic acid molecule encoding reuterin which is anantimicrobial substance effective, inter alia, against Streptococcusmutans. Reuterin is, for example, described in Talarico et al.,Chemother. 33 (1989), 674-679.

A mutant of the binder microorganism belonging to the group of lacticacid bacteria, preferably a mutant of the deposited Lactobacillusstrains, is preferably artificially mutated. In accordance with thepresent invention, the term “mutated” means one or more permanentmodifications of genetic material, i.e. nucleic acids, caused, forexample, naturally or by physical means or chemicalcompounds/substances/agents, such as EMS or ENU. Said modificationsinclude point mutations, like transitions or transversions,deletion/insertion/addition of one or more bases within a nucleicacid/gene/chromosome thereby modifying the nucleic acid/gene/chromosomewhich can cause, inter alia, aberrant geneexpression/transcription/translation or inactive gene products,constitutive active/inactive gene products leading to e.g.dominant-negative effects. Preferably, a mutation leads to in increasedcapability of specifically binding mutans Streptococci. Thus, it is alsopreferred that the mutant cells of the deposited microorganism whichharbour one or more mutations in one or more desired genes or in whichone or more mutations in one or more desired genes is induced by methodsknown to the person skilled in the art. It is also known in the priorart that mutated or genetically engineered bacterial cells can beselected by any suitable method/phenotype. In the context of the presentinvention, a mutant having an increased capability to specifically bindto mutans Streptococci can be tested in accordance with the methodsdescribed above. The term “mutant”, however, also includes cells ofabove mentioned binder microorganism belonging to the group of lacticacid bacteria, preferably cells of the deposited microorganism, whichharbour naturally-occurring, spontaneous mutations in their genome, i.e.bacterial chromosome. “Spontaneous mutations” are mutations that arisenaturally, i.e., without direct genetic manipulation by man, or byexposure to a mutagen. Selection of spontaneous mutants can beaccomplished by culturing the strain and selecting the desired variantsby, for example, the variant bacterium's capability to show an improvedbinding to mutans Streptococci. Methods for selection of spontaneousmutants are well known in the art (see, for example, Sambrook, Russell“Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory,N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, GreenPublishing Associates and Wiley Interscience, N.Y. (1989)). For example,such mutations may occur during cultivation, for example, during thenormal cell division process coupled with DNA replication or duringpassaging and/or preserving the mutant of the above mentioned bindermicroorganism belonging to the group of lactic acid bacteria.

However, even though genetic manipulation of microorganisms may impartbeneficial or even highly beneficial properties to the microorganisms,it is alternatively preferred that the binder microorganism andpreferably all microorganisms used according to the present invention isnot a genetically modified microorganism as defined in Article 2(2) ofDirective 2001/18/EC in the respective version applicable at the filingday of this application. However, microorganisms obtained through thetechniques of genetic modification listed in Annex 1B to said Directive2001/18/EC according to the invention are preferably not considered agenetically modified microorganism as defined in Article 2 (2) ofDirective 2001/18/EC. By excluding genetically modified microorganisms,rational and irrational fears of consumers can be avoided, therebyincreasing consumer compliance with oral healthcare instructions.

According to the present invention the binder microorganism, preferablyof the Lactobacillaceae family and even more preferably of genuslactobacillus, most preferred one of the above mentioned particularlypreferred strains, does not comprise genetic material which has beenaltered in a way that does not occur naturally by mating and/or naturalrecombination. Such techniques include

-   (1) recombinant nucleic acid techniques involving the formation of    new combinations of genetic material by the insertion of nucleic    acid molecules produced by whatever means outside an organism, into    any virus, bacterial, plasmid or other vector system and their    incorporation into a host organism in which they do not naturally    occur but in which they are capable of continued propagation;-   (2) techniques involving the direct introduction into an organism of    heritable material prepared outside the organism including    micro-injection, macro-injection and micro-encapsulation;-   (3) cell fusion including protoplast fusion or hybridization    techniques where life cells with new combinations of heritable    material are formed through the fusion of two or more cells by means    of methods that do not occur naturally.

Natural processes such as conjugation, transduction and transformation,however, are preferably not excluded according to the present invention.Further preferably not excluded techniques according to the presentinvention are, on the condition that they do not involve the use ofrecombinant nucleic acid molecules or genetically modified organismsother than those produced by one or more of the techniques/methodslisted hereinafter are

-   (1) mutagenesis by spontaneous or induced spontaneous mutation, and-   (2) cell fusion including protoplast fusion of microorganisms which    can exchange genetic material through traditional breading methods.

According to the invention, it is thus preferred that the bindermicroorganism and preferably also any other microorganism used accordingto the present invention does not comprise genetic material of anorganism of super kingdom archaea or eukaryota, of course with theexception of such genetic material which can naturally be found instrains of the same species or, less preferably, at least in the samegenus as the microorganism used according to the present invention.Further preferably, the microorganism used according to the presentinvention does not comprise genetic material of a microorganism foundonly in a phylum other than firmicutes and even more preferably does notcomprise genetic material found only in or taken from microorganisms ofa class other than bacilli, even more preferably does not comprisegenetic material only found in or taken from microorganisms of an orderother than Lactobacillales.

Where the microorganism used according to the present invention andparticularly the binder microorganism is of family Lactobacillaceae,then it is preferred that the respective microorganism does not comprisegenetic material found only in or taken from microorganisms of a familyother than Lactobacillaceae. Even more preferably, if the microorganismof the present invention and particularly the binder microorganism is ofgenus lactobacillus, then the respective microorganism does not comprisegenetic material found only in or taken from microorganisms of a genusother than lactobacillus.

The composition according to the invention preferably is preferably ananticariogenic food or feed composition, or an anticariogenicpharmaceutical composition. The present invention thus relates to theuse of the above mentioned binder microorganism or fragment thereof forthe preparation of an anticariogenic composition, preferably apharmaceutical or cosmetic composition, for the treatment or preventionof caries caused by mutans Streptococci and/or Streptococcus mutans.

The term “composition”, as used in accordance with the presentinvention, indicates to compositions which comprise at least one bindermicroorganism—possibly in thermally inactivated or lyophilized form—orfragment thereof, preferably a deposited microorganism as describedabove—possibly in thermally inactivated or lyophilized form—or afragment of said microorganism. It is envisaged that the compositions asused in accordance with the present invention comprise theaforementioned ingredients in any combination. It may, optionally,comprise at least one further ingredient suitable for preventing and/ortreating caries. Accordingly, it may optionally comprise any combinationof the hereinafter described further ingredients. The term “ingredientssuitable for preventing and/or treating caries” encompasses compounds orcompositions and/or combinations thereof which either inhibit thebinding of mutans Streptococci to the surface of teeth, to pelliclesand/or which inactivate mutans Streptococci. More preferably, said termencompasses compounds or compositions and/or combinations thereof whichmay inhibit the adhesion of mutans Streptococci to the surface of teeth,inhibit the activity of glycosyltransferases of mutans Streptococci,inhibit or inactivate mutans Streptococci, inhibit theagglutinin-dependent binding of mutans Streptococci and/or inhibit thesaccharose-dependent binding of mutans Streptococci as will be describedbelow.

The composition of the present invention preferably is a composition fororal health of a pet animal, and even more preferably is a compositionfor prevention or reduction of dental calculus. Such compositions areknown for example from EP 01 41 645 A2, WO 0150882 A2, WO 2001/070043A2, WO 02/078462 A1, WO 2004/082518 A2, WO 2005/092087 A2 and WO2010/052467 A2. The foods and feeds described in these documents andparticularly in the examples mentioned in these documents areincorporated herein as examples of preferred base compositions. Thesebase compositions according to the invention are further amended byincorporating a binder microorganism of the present invention orfragment thereof.

It is a particular advantage of the present invention that by the actionof a binder microorganism or fragment thereof, i.e. by specificallybinding to a mutans Streptococcus and most preferably by specificallybinding Streptococcus mutans, the build-up of dental calculus can bedelayed or slowed down without having to rely on the action ofbactericidal agents or other chemical agents which would killmicroorganisms in the oral cavity. It is suspected that the normal oralmicroflora is beneficial for humans and animals, for example as suchmicroorganisms of the normal microflora will compete with pathogens fornutrition, thereby limiting the growth of pathogens and avoidinginfections. The present invention allows to let the normal oralmicroflora remain largely undisturbed and still confers oral careproperties, preferably the prevention or slow down of dental calculusformation.

Another advantage of the present invention in the prophylaxis andtreatment of dental calculus is that the present invention does notrequire the presence of decalcifying agents like zinc sulfate, solublepyrophosphates, sodium tripolyphosphate and soluble diphosphonates. Suchagents sequester calcium from the oral cavity, thereby removing a keycomponent of dental calculus formation. However, tooth enamel is largelymade up of hydroxyl apatite, which is a mineral with high calciumcontent. Thus, removal of calcium from the oral cavity is implicated infurther weakening of teeth, which is undesired. The present invention,on the other hand, allows to treat or prevent formation of dentalcalculus without sequestering calcium which would be required formaintenance of healthy tooth enamel.

For best results in the prevention of dental calculus formation, acomposition of the present invention therefore comprises a bindermicroorganism or fragment thereof, wherein the binder microorganismpreferably is of family Lactobacillaceae, more preferably of genusLactobacillus and most preferably is one of the above mentioneddeposited strains of binder microorganisms, and further comprisesdecalcifying agents in a concentration such that binding of bindermicroorganisms or fragments thereof to mutans Streptococci is reduced byat most 10% as determined by nephelometry, and preferably does notcomprise such agents. In such preferred compositions, decalcifyingagents preferably are selected from the group of zinc sulfate, zincchloride, soluble pyrophosphates, sodium tripolyphosphate and solublediphosphonates.

The present invention also provides the use of a binder microorganism orfragment thereof as described above for the preparation of a medicamentfor prevention or treatment of dental calculus, preferably in a child orin a pet animal. As indicated above, the binder microorganism can alsobe in a thermally inactivated form, particularly in an autoclaved form,or in a lyophilized form. The binder microorganism preferably is of thefamily of Lactobacillaceae, even more preferably of genus Lactobacillus,even more preferably of species Lactobacillus paracasei or Lactobacillusrhamnosus. Particularly, the binder microorganism of the presentinvention can be selected from the above indicated strains of L.paracasei or L. rhamnosus, respectively, having any DSMZ accessionnumber of 16667 to 16673, or a mutant or derivative thereof.

Most preferably, the composition according to the present invention is afood or feed composition, particularly for children or pet animals. Suchoral health food or feed compositions are sometimes termed functionalfood or functional feed. The terms “food” and “feed” are used accordingto the invention regardless of the nutritional value of correspondingcompositions and are thus not limited to particular nutritionalpurposes, even though food and feed compositions according to thepresent invention can be tailored to such particular purposes. The terms“food” and “feed” thus indicate that the respective composition issuitable for being placed in the oral cavity and for ingestion.

Of particular importance according to the present invention are food orfeed compositions for animals, preferably for pet animals, and mostpreferably for cats, dogs, rats, hamsters and guinea pigs. In suchanimals, the formation of caries is particularly notorious and alsodifficult to treat, as any dental treatment like removal of dentalcalculus requires anaesthesis of the pet. The present invention is thusparticularly suitable for preventing the need for such stressfultreatment.

The feed composition of the present invention preferably is a pet feed,i.e. a composition for an animal fed, bred or kept, but not normallyused for human consumption in the European Community. Preferably, “pet”according to the present invention is a mammal of order carnivora, evenmore preferably of suborder caniformia or suborder feliformia, and mostpreferably of the canidae or felidae family. Further preferred pets areof the order rodentia, wherein particularly preferred animals are mice,rabbits, hamsters and guinea pigs.

For pet feed it is preferred that the compound of the present inventionis a compound feed, a complete feed, a complementary feed or a mineralfeed. According to the present invention, the term “compound feed” meansa mixture of at least two feed materials, whether or not containing feedadditives, for oral animal feeding in the form of complete orcomplementary feed. The term “complete feed” means compound feed which,by reason of its composition, is sufficient for a daily ration. The term“complementary feed” according to the invention means compound feedwhich has a high content of certain substances but which, by reason ofits composition, is sufficient for a daily ration only if used incombination with other feed. The term “mineral feed” means complementaryfeed containing at least 40% crude ash. Finally, the term “feedmaterial” according to the invention means products of a vegetable oranimal origin, whose principal purpose is to meet animal nutricialmeads, in their natural state, fresh state or preserved, and products,derived from the industrial processing thereof, and organic or inorganicsubstances, whether or not containing feed additives, which are intendedfor use in oral animal feeding either directly as such or afterprocessing, or in the preparation of compound feed, or as carrier ofpre-mixtures. The term “oral animal feeding” means the introduction offeed into an animal gastrointestinal tract through the mouth with theaim of meeting the animal's nutritional needs and/or maintaining theproductivity of normally healthy animals.

According to the present invention, the composition comprising thebinder microorganism can also be a oral health composition. Preferredexamples of oral care compositions according to the present inventionare tooth paste, dentrifices, tooth powders, topical oral jelly, mouthrinses, denture products, mouth sprays, lozenges, oral tablets, chewinggum, dental floss or dental tape, and particularly for animals, chewproducts.

A preferred oral care composition according to the present inventioncomprises not only the binder microorganism or fragment thereof, butalso an orally acceptable carrier, such carrier can be any suitablevehicle which can be applied to the oral cavity in a safe and effectivemanner, such that the binder microorganism of the present inventionand/or the fragment thereof can bind to one or more strains of mutansStreptococci and preferably to one or more strains of Streptococcusmutans, thus exerting the anti-dental calculus and/or anti-cariogenicand/or anti-oral-malodor effect. The oral care composition may be asingle or multiple phase composition.

In particular, the dentrifice of the present invention can be a paste,gel, or liquid formulation unless otherwise specified. The dentifricecomposition may be in any desired form, such as deep striped, surfacestriped, multilayered, having the gel surrounding the paste, or anycombination thereof. The dentifrice composition may be contained in aphysically separated compartment of a dispenser and dispensedside-by-side. Dentifrice compositions are, for example, described in EP0 617 608 B1.

Preferred dentifrice compositions are described in Examples 21 to 24 ofWO 2008/074473 A2. In addition to the above described components, thedentifrice compositions of this invention can contain a variety ofoptional dentifrice ingredients some of which are described below.Optional ingredients include, for example, but are not limited to,adhesives, sudsing agents, flavouring agents, sweetening agents,additional antiplaque agents, additional abrasives, and colouringagents. These and other optional components are further described, forexample, in U.S. Pat. No. 5,004,597, U.S. Pat. No. 4,885,155, U.S. Pat.No. 3,959,458 and U.S. Pat. No. 3,937,807.

For example, the toothpaste may include one or more surfactants,chelating agents, fluoride sources, teeth whitening actives and teethcolor modifying substances, thickening agents, humectants, flavouringand sweetening agents, alkali metal bicarbonate salt, miscellaneouscarriers and/or other active agents.

One of the preferred optional agents as used in accordance with thepresent invention is a surfactant, preferably one selected from thegroup consisting of sarcosinate surfactants, isethionate surfactants andtaurate surfactants. Preferred for use herein are alkali metal orammonium salts of these surfactants. Most preferred herein are thesodium and potassium salts of the following: lauroyl sarcosinate,myristoyl sarcosinate, palmitoyl sarcosinate, stearoyl sarcosinate andoleoyl sarcosinate.

Another preferred optional agent is a chelating agent such as tartaricacid and pharmaceutically-acceptable salts thereof, citric acid andalkali metal citrates and mixtures thereof. Chelating agents are able tocomplex calcium found in the cell walls of the bacteria. Chelatingagents can also disrupt plaque by removing calcium from the calciumbridges, which help hold this biomass intact. However, as indicatedabove there is a preferred maximum concentration of such agents or suchcompositions which are intended for a particularly gentle treatment ofteeth.

It is common to have an additional water-soluble fluoride compoundpresent in dentifrices and other oral compositions in an amountsufficient to give a fluoride ion concentration in the. composition at25° C., and/or when it is used of from about 0.0025% to about 5.0% byweight, preferably from about 0.005% to about 2.0% by weight, to provideadditional anticaries effectiveness. A wide variety of fluorideion-yielding materials can be employed as sources of soluble fluoride inthe present compositions. Examples of suitable fluoride ion-yieldingmaterials are found in U.S. Pat. No. 3,535,421 and U.S. Pat. No.3,678,154. Representative fluoride ion sources include stannousfluoride, sodium fluoride, potassium fluoride, sodiummonofluorophosphate and many others. Stannous fluoride and sodiumfluoride are particularly preferred, as well as mixtures thereof.

The oral care compositions according to the present invention may alsocomprise teeth whitening actives, including bleaching or oxidizingagents such as peroxides, perborates, percarbonates, peroxyacids,persulfates, metal chlorites, and combinations thereof. Suitableperoxide compounds include hydrogen peroxide, urea peroxide, calciumperoxide, and mixtures thereof. A preferred percarbonate is sodiumpercarbonate. Other suitable whitening agents include potassium,ammonium, sodium and lithium persulfates and perborate mono- andtetrahydrates, and sodium pyrophosphate peroxyhydrate. Suitable metalchlorites include calcium chlorite, barium chlorite, magnesium chlorite,lithium chlorite, sodium chlorite, and potassium chlorite. The preferredchlorite is sodium chlorite. Additional whitening actives may behypochlorite and chlorine dioxide.

In addition to bleaching agents as teeth whitening agents, teeth colormodifying substances may be considered among the oral care activesuseful in the present invention. These substances are suitable formodifying the color of the teeth to satisfy the consumer. Thesesubstances comprise particles that when applied on the tooth surfacemodify that surface in terms of absorption and, or reflection of light.Such particles provide an appearance benefit when a film containing suchparticles is applied over the surfaces of a tooth or teeth.

In preparing toothpaste or gels, it is necessary to add some thickeningmaterial to provide a desirable consistency of the composition, toprovide desirable active release characteristics upon use, to provideshelf stability, and to provide stability of the composition, etc.

Preferred thickening agents are carboxyvinyl polymers, carrageenan,hydroxyethyl cellulose, laponite and water soluble salts of celluloseethers such as sodium carboxymethylcellulose and sodium carboxymethylhydroxyethyl cellulose. Natural gums such as gum karaya, xanthan gum,gum arabic, and gum tragacanth can also be used. Colloidal magnesiumaluminum silicate or finely divided silica can be used as part of thethickening agent to further improve texture.

Another optional component of the topical, oral carriers of thecompositions of the subject invention is a humectant. The humectantserves to keep toothpaste compositions from hardening upon exposure toair, to give compositions a moist feel to the mouth, and, for particularhumectants, to impart desirable sweetness of flavor to toothpastecompositions.

The humectant, on a pure humectant basis, generally comprises from about0% to about 70%, preferably from about 5% to about 25%, by weight of thecompositions herein. Suitable humectants for use in compositions of thesubject invention include edible polyhydric alcohols such as glycerin,sorbitol, xylitol, butylene glycol, polyethylene glycol, and propyleneglycol, especially sorbitol and glycerin.

Flavoring and sweetening agents can also be added to the compositions.Suitable flavoring agents include oil of wintergreen, oil of peppermint,oil of spearmint, clove bud oil, menthol, anethole, methyl salicylate,eucalyptol, cassia, 1-menthyl acetate, sage, eugenol, parsley oil,oxanone, alpha-irisone, marjoram, lemon, orange, propenyl guaethol,cinnamon, vanillin, thymol, linalool, cinnamaldehyde glycerol acetalknown as CGA, and mixtures thereof. Flavouring agents are generally usedin the compositions at levels of from about 0.001% to about 5%, byweight of the composition.

Sweetening agents which can be used include sucrose, glucose, saccharin,dextrose, levulose, lactose as described herein above, mannitol,sorbitol, fructose, maltose, xylitol, saccharin salts, thaumatin,aspartame, D-tryptophane, dihydrochalcones, acesulfame and cyclamatesalts, especially sodium cyclamate and sodium saccharin, and mixturesthereof. An infant food composition preferably contains from about 0.1%to about 10% of these agents, preferably from about 0.1% to about 1%, byweight of the composition. Preferably, a pet food according to thepresent invention only has a low content of such sugars which can bemetabolized by mutans Streptococci and preferably Streptococcus mutans,to avoid cariogenic activity of mutans Streptococci. Thus, the contentof sucrose, cane sugar, caramel, corn syrup, corn molasses, glucose,fructose and sorbitol is preferably kept low, with a maximum content of20 wt.-% of the total infant or pet food being preferred; a maximumcontent of 5 wt.-% is even more preferred, and a maximum content of 2wt.-% being most preferred. Also preferably, the content of each ofsaccharin, dextrose, levulose, lactose, mannitol, maltose and xylitol isless than 20 wt.-%, more preferably less than 5 wt.-% and mostpreferably less than 2 wt.-%, with xylitol preferably being notcontained in the pet food at all.

The oral care composition of the present invention may also include analkali metal bicarbonate salt. Alkali metal bicarbonate salts aresoluble in water and unless stabilized, tend to release carbon dioxidein an aqueous system. Sodium bicarbonate, also known as baking soda, isthe preferred alkali metal bicarbonate salt. The present composition maycontain from about 0.5% to about 30%, preferably from about 0.5% toabout 15%, and most preferably from about 0.5% to about 5% of an alkalimetal bicarbonate salt. Water employed in the preparation ofcommercially suitable oral compositions should preferably be of low ioncontent and free of organic impurities. Water generally comprises fromabout 10% to about 50%, and preferably from about 20% to about 40%, byweight of the aqueous toothpaste compositions herein. These amounts ofwater include the free water which is added plus that which isintroduced with other materials, such as with sorbitol. Titanium dioxidemay also be added to the present composition. Titanium dioxide is awhite powder, which adds opacity to the compositions. Titanium dioxidegenerally comprises from about 0.25% to about 5% by weight of thedentifrice compositions.

The pH of the present compositions is preferably adjusted through theuse of buffering agents. Buffering agents, as used herein, refer toagents that can be used to adjust the pH of the compositions to a rangeof about 4.5 to about 9.5, preferably 4.5 to 8.5. Buffering agentsinclude monosodium phosphate, trisodium phosphate, sodium hydroxide,sodium carbonate, sodium acid pyrophosphate, citric acid, and sodiumcitrate. Buffering agents can be administered at a level of from about0.5% to about 10%, by weight of the present compositions. The pH ofdentifrice compositions is measured from a 3:1 aqueous slurry ofdentifrice, e.g., 3 parts water to 1 part toothpaste.

Other optional agents that may be used in the present compositionsinclude dimethicone copolyols selected from alkyl- andalkoxy-dimethicone copolyols, such as C12 to C20 alkyl dimethiconecopolyols and mixtures thereof. Highly preferred is cetyl dimethiconecopolyol marketed under the Trade Name Abil EM90. The dimethiconecopolyol is generally present in a level of from about 0.01% to about25%, preferably from about 0.1% to about 5%, more preferably from about0.5% to about 1.5% by weight. The dimethicone copolyols aid in providingpositive tooth feel benefits. Other useful carriers include biphasicdentifrice formulations such as those disclosed in U.S. Pat. No.5,213,790, U.S. Pat. No. 5,145,666, U.S. Pat. No. 5,281,410, U.S. Pat.No. 4,849,213 and U.S. Pat. No. 4,528,180.

The present compositions may also include other active agents, such asantimicrobial agents. Included among such agents are water insolublenon-cationic antimicrobial agents such as halogenated diphenyl ethers,phenolic compounds including phenol and its homologs, mono andpoly-alkyl and aromatic halophenols, resorcinol and its derivatives,bisphenolic compounds and halogenated salicylanilides, benzoic esters,and halogenated carbanilides. The water soluble antimicrobials includequaternary ammonium salts and bis-biquanide salts, among others.Triclosan monophosphate is an additional water soluble antimicrobialagent. The quaternary ammonium agents include those in which one or twoof the substitutes on the quaternary nitrogen has a carbon chain length(typically alkyl group) from about 8 to about 20, typically from about10 to about 18 carbon atoms while the remaining substitutes (typicallyalkyl or benzyl group) have a lower number of carbon atoms, such as fromabout 1 to about 7 carbon atoms, typically methyl or ethyl groups.Dodecyl trimethyl ammonium bromide, tetradecylpyridinium chloride,domiphen bromide, N-tetradecyl-4-ethyl pyridinium chloride, dodecyldimethyl (2-phenoxyethyl) ammonium bromide, benzyl dimethylstearylammonium chloride, cetyl pyridinium chloride, quaternized5-amino-1,3-bis(2-ethyl-hexyl)-5-methyl hexa hydropyrimidine,benzalkonium chloride, benzethonium chloride and methyl benzethoniumchloride are exemplary of typical quaternary ammonium antibacterialagents. Other compounds are bis[4-(R-amino)-1-pyridinium] alkanes asdisclosed in U.S. Pat. No. 4,206,215. Other antimicrobials such ascopper bisglycinate, copper glysinate, zinc citrate, and zinc lactatemay also be included. Enzymes are another type of active that may beused in the present compositions. Useful enzymes include those thatbelong to the category of proteases, lytic enzymes, plaque matrixinhibitors and oxidases: Proteases include papain, pepsin, trypsin,ficin, bromelin; cell wall lytic enzymes include lysozyme; plaque matrixinhibitors include dextranses, mutanases; and oxidases include glucoseoxidase, lactate oxidase, galactose oxidase, uric acid oxidase,peroxidases including horse radish peroxidase, myeloperoxidase,lactoperoxidase, chloroperoxidase. The oxidases also havewhitening/cleaning activity, in addition to anti-microbial properties.Such agents are disclosed in U.S. Pat. No. 2,946,725 and in U.S. Pat.No. 4,051,234. Other antimicrobial agents include chlorhexidine,triclosan, triclosan monophosphate, and flavor oils such as thymol.Triclosan and other agents of this type are disclosed in U.S. Pat. No.5,015,466 and U.S. Pat. No. 4,894,220. These agents, which provideanti-plaque benefits, may be present at levels of from about 0.01% toabout 5.0%, by weight of the dentifrice composition.

The term “chewing gum” as defined herein means a confectionerycomposition which is suitable for chewing and which comprises anysuitable amount of elastomer, known to the person skilled in the art,preferably an amount of 2% or greater, by weight of the composition.Suitable lozenge and chewing gum components are, for example, disclosedin U.S. Pat. No. 4,083,955, U.S. Pat. No. 6,770,264 or U.S. Pat. No.6,270,781. Preferred lozenges are those described in Examples 19 and 20of WO 2008/074473 A2. A preferred chewing gum composition is describedin Example 25 of WO 2008/074473 A2.

Compositions of the present invention preferably comprise an elastomer,or mixture of several different elastomers. Elastomeric materials aregenerally known in the art but illustrative examples includestyrene-butadiene rubber (SBR); synthetic gums; polyisobutylene andisobutylene-isoprene copolymers; natural gums; chicle; natural rubber;jelutong; balata; guttapercha; lechi caspi; sorva; and mixtures thereof.Compositions as used in accordance with the present invention preferablycomprise from about 2% to about 30%, more preferably from about 5% toabout 25%, by weight, of elastomer. These levels are determined by thedesired final texture of the chewing gum since when the total level ofelastomer is below about 2% the base composition lacks elasticity,chewing texture, and cohesiveness whereas at levels above about 30% theformulation is hard, rubbery and maintains a tight chew. Elastomersolvents are also preferably present in compositions as used inaccordance with the present invention since they aid softening of theelastomer component. Preferred examples of elastomer solvents for useherein include the pentaerythritol ester of partially hydrogenated woodrosin, pentaerythritol ester of wood rosin, glycerol ester of partiallydimerized rosin, glycerol ester of polymerised rosin, glycerol ester oftall oil, wood or gum rosin, glycerol ester of partially hydrogenatedrosin, methyl ester of partially hydrogenated rosin, and mixturesthereof. Compositions as used in accordance with the present inventionpreferably comprise from about 2% to about 50%, more preferably fromabout 10% to about 35%, by weight, of elastomer solvent.

Lozenges of this invention can be prepared, for example, byart-recognized techniques for forming compressed tablets where thedisaccharide is dispersed on a compressible solid carrier, optionallycombined with any appropriate tableting aids such as a lubricant (e.g.,magnesium-stearate) and is compressed into tablets. The solid carriercomponent for such tableting formulations can be a saliva-soluble solid,such as a cold water-soluble starch or a monosaccharide, so that thelozenge will readily dissolve in the mouth to release the containeddisaccharide acid in saliva solution for contact with and absorption bythe oral/pharyngeal mucosa when the lozenge is held in the mouth. The pHof the above-described formulations can range from about 4 to about 8.5.Lozenges in accordance with the present invention can also be preparedutilizing other art-recognized solid unitary dosage formulationtechniques.

A mouth wash or mouth rinse of the present invention could contain EtOHor could be EtOH-free, and could contain other active ingredients, ase.g. antimicrobials such as Chlorhexidin. A preferred mouth wash ormouth rinse of the present invention could be as follows:

A Olium menthae 1.2 parts

-   -   Tinctura Arnicae 3.0 parts    -   Tinctura Myrrhae 3.0 parts    -   Tween 5.0 parts

B Spiritus 90% 50.0 parts

C Sodium Benzoate 0.2 parts

Sweetening agent (e.g. aspartame) 0.02 parts

Aqua destilata ad 100.0

A is to be well mixed, B is added under stirring and C is addedsubsequently. The resulting clear liquid is to be filtered within 48hours after preparation. Another preferred mouth wash is described inExample 26 of WO 2008074473 A2.

Regardless of the dosage form, liquid or solid, in one preferredembodiment of the present invention the dosage form is held in theconsumers mouth, preferably the pet animal's mouth, for a period of timeto promote contact of the microorganism or analog or fragment of a abovementioned microorganism belonging to the group of lactic acid bacteriawith the patient's oral cavity.

The terms “dental floss” and “dental tape” as used herein refer to amaterial to dislodge and remove decomposing food material thataccumulated at interproximal and subgingival surfaces and to dislodgeand remove bacteria, plaque and/or calculus that accumulated in the oralcavity. The dental floss or dental tape may further contain, in additionto the microorganisms according to the present invention as describedherein, cleaners, abrasives, tartar control ingredients, whiteners,surfactants and/or active ingredients like fluorides, antimicrobials,chemotherapeutic agents or antibiotics. Further additional agents areantiplaque agents, flavouring agents and colouring agents. The dentalfloss or dental tape may be in any suitable form, known to the personskilled in the art, for example, in the form of PTFE (Teflon) dentalflosses as described, for instance, in U.S. Pat. No. 3,664,915, U.S.Pat. No. 3,953,566, U.S. Pat. No. 3,962,153, U.S. Pat. No. 4,096,227,U.S. Pat. No. 4,187,390, U.S. Pat. No. 4,256,806, U.S. Pat. No.4,385,093, U.S. Pat. No. 4,478,665, U.S. Pat. No. 4,776,358, U.S. Pat.No. 5,033,488, U.S. Pat. No. 5,209,251, U.S. Pat. No. 5,220,932, U.S.Pat. No. 5,518,012, U.S. Pat. No. 5,718,251, U.S. Pat. No. 5,765,576 orU.S. Pat. No. 5,911,228, in the form of monofilament interproximaldevices as described, for instance, in U.S. Pat. No. 3,800,812, U.S.Pat. No. 4,974,615, U.S. Pat. No. 5,760,117, U.S. Pat. No. 5,433,226,U.S. Pat. No. 5,479,952, U.S. Pat. No. 5,503,842, U.S. Pat. No.5,755,243, U.S. Pat. No. 5,884,639, U.S. Pat. No. 6,003,525 or U.S. Pat.No. 6,027,592, or in the form of biocomponent tapes. Preferably, thedental floss or dental tape may be in the form of an elastomeric coatedmonofilament as described, for instance, in US 20050226820 or in theform of an oriented thermoplastic based dental tape as described, forinstance, in US 20020144704.

The oral care cosmetic compositions as described herein, particularlyanti-dental calculus compositions, anti-caries compositions andanti-oral malodor compositions, may be used in the ambit of human oraladministration as well as in the ambit of veterinary oraladministration, preferably for non-human mammals, more preferably forpets. If the composition is used in the ambit of veterinary oraladministration, the composition may contain further ingredients suitablefor such an administration, as known by a person skilled in the art.

A pharmaceutical composition according to the present inventionpreferably further comprises a pharmaceutical acceptable carrier orexcipient.

Pharmaceutical compositions comprise a therapeutically effective amountthe above mentioned microorganism or fragment thereof and can beformulated in various forms, e.g. in solid, liquid, powder, aqueous,lyophilized form.

The pharmaceutical composition may be administered with apharmaceutically acceptable carrier to a patient, preferably a humanbeing or an animal, and most preferably a child or pet animal. The term“pharmaceutically acceptable” means approved by a regulatory agency orother generally recognized pharmacopoeia for use in animals, and moreparticularly in humans. A preferred pharmaceutical composition as usedin accordance with the present invention does not contain lactose in arange between 1% (w/w) and 6% (w/w). It is also preferred that thepharmaceutical composition contains not more than 1% (w/w) lactose, e.g.it contains less than 1%, preferably less than 0.9% (w/w), 0.8% (w/w)lactose, etc. or that the pharmaceutical composition contains more than6%, 7%, 8% etc. (w/w) lactose. Alternatively, but also preferred is thatthe pharmaceutical composition does not contain lactose.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such a carrier ispharmaceutically acceptable, i.e. is non-toxic to a recipient at thedosage and concentration employed. It is preferably isotonic, hypotonicor weakly hypertonic and has a relatively low ionic strength, such asprovided by a sucrose solution. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumion, dried skimmed milk, glycerol, propylene, glycol, water, ethanol andthe like. The excipient may contain lactose as described herein above,most preferably it is lactose-free. The composition, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. Oral formulation caninclude standard carriers such as pharmaceutical grades of maltodextrin,mannitol, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E.W. Martin.Skimmed milk, skimmed milk powder, non-milk or non-lactose containingproducts may also be employed. The skimmed milk powder is conventionallysuspended in phosphate buffered saline (PBS), autoclaved or filtered toeradicate proteinaceous and living contaminants, then freeze dried heatdried, vacuum dried, or lyophilized. Some other examples of substanceswhich can serve as pharmaceutical carriers are sugars, such as glucoseand sucrose; starches such as corn starch and potato starch; celluloseand its derivatives such as sodium carboxymethycellulose, ethylcelluloseand cellulose acetates; powdered tragancanth; malt; gelatin; talc;stearic acids; magnesium stearate; calcium sulfate; calcium carbonate;vegetable oils, such as peanut oils, cotton seed oil, sesame oil, oliveoil, corn oil and oil of theobroma; polyols such as propylene glycol,glycerine, sorbitol, manitol, and polyethylene glycol; agar; alginicacids; pyrogen-free water; isotonic saline; cranberry, extracts andphosphate buffer solution; skim milk powder; as well as other non-toxiccompatible substances used in pharmaceutical formulations such asVitamin C, estrogen and echinacea, for example. Wetting agents andlubricants such as sodium lauryl sulfate, as well as colouring agents,flavouring agents, lubricants, excipients, tabletting agents,stabilizers, anti-oxidants and preservatives, can also be present.

Preferably, the oral formulation contains lactose as described hereinand is most preferably lactose-free. Various carriers and/or excipientssuitable for oral administration which are well known in the art may beused for the purpose of this invention. The non-cariogenic compositionmay, if desired, further contain various known additives such as, forexample, preservatives, hardening agents, lubricants, emulsifiers,stabilizers, essence and the like. Such compositions will contain atherapeutically effective amount of the aforementioned compounds,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

A preferred composition of the present invention does not containlactose in a range between 1% (w/w) and 6% (w/w). It is also preferredthat the composition contains not more than 1% (w/w) lactose, e.g. itcontains less than 1%, preferably less than 0.9% (w/w), 0.8% (w/w)lactose, etc. or that the composition contains more than 6%, 7%, 8% etc.(w/w) lactose. Alternatively, but also preferred is that the compositiondoes not contain lactose.

In a further aspect, a composition of the present invention may beproduced by comprising the steps of formulating a binder microorganismfragment thereof with a cosmetically, orally or pharmaceuticalacceptable carrier or excipient. Preferably, this microorganism is adeposited microorganism as described herein above or a mutant,derivative, analog or fragment thereof. A preferred composition inaccordance with the present invention does not contain lactose in arange between 1% (w/w) and 6% (w/w). It is also preferred that thecomposition contains not more than 1% (w/w) lactose, e.g. it containsless than 1%, preferably less than 0.9% (w/w), 0.8% (w/w) lactose, etc.,or that the composition contains more than 6%, 7%, 8% etc. (w/w)lactose. Alternatively, but also preferred is that the composition doesnot contain lactose.

The composition of the present invention, preferably a food or feedcomposition including pharmaceutical compositions, comprises a bindermicroorganism as described above, potentially in a thermally inactivatedor lyphilized form, in an amount of 10² to 10¹² cells, preferably 10³ to10⁸ cells per mg in a solid form of the composition. In case of a liquidform of the composition, the amount of the microorganisms is 10² to 10¹³cells per ml.

However, for specific compositions the amount of the microorganism maybe different as is described herein.

Preferably, the concentration of binder microorganisms in thecomposition of the present invention is 0.01 wt.-% to 10 wt.-%, relativeto the total mass of the composition. Even more preferably, theconcentration of binder microorganisms in the composition of the presentinvention is 0.025 wt.-% to 2 wt.-%. As described above, when fragmentsare used instead of binder cells, then the concentration of fragments ischosen to be the same as the peptidoglycan content of binder cells.

In accordance with the present invention, the term food encompasses alleatable and drinkable foods and drinks. Accordingly, the microorganismor fragment thereof may be included in a food or drink. These are, forexample, gum, spray, beverage, candies, infant formula, ice cream,frozen dessert, sweet salad dressing, milk preparations, cheese, quark,lactose-free yogurt, acidified milk, coffee cream or whipped cream andthe like.

Milk-based products are envisaged within the framework of the invention.Milk is however understood to mean that of animal origin, such as cow,goat, sheep, buffalo, zebra, horse, donkey, or camel, and the like. Themilk may be in the native state, a reconstituted milk, a skimmed milk ora milk supplemented with compounds necessary for the growth of thebacteria or for the subsequent processing of fermented milk, such asfat, proteins of a yeast extract, peptone and/or a surfactant, forexample. The term milk also applies to what is commonly called vegetablemilk, that is to say extracts of plant material which have been treatedor otherwise, such as leguminous plants (soya bean, chick pea, lentiland the like) or oilseeds (colza, soya bean, sesame, cotton and thelike), which extract contains proteins in solution or in colloidalsuspension, which are coagulable by chemical action, by acidfermentation and/or by heat. Finally, the word milk also denotesmixtures of animal milks and of vegetable milks.

Where the binder microorganism of this invention or fragment thereof areadded to yogurt and the like having similar contents, it is sufficientto add the microorganism of this invention at a concentration of about10⁵-10⁷ cells/ml, or the equivalent amount of fragment thereof. In sucha case, it is possible to completely prevent or inhibit formation of abiofilm by mutans Streptococci, preferably by Streptococcus mutans, andthus to prevent or slow the development of dental calculus, oralmalodour or dental caries induced by cariogenic strains, withoutsignificant side effect upon the quality of the drink per se.

Such food drink or feed can be produced by a general method forproducing foods and drinks or feeds, including adding the activeingredient to a raw or cooked material of the food, drink or feed. Thefood, drink or feed in accordance with the present invention can bemolded and granulated in the same manner as generally used for foods,drinks or feeds. The molding and granulating method includes granulationmethods such as fluid layer granulation, agitation granulation,extrusion granulation, rolling granulation, gas stream granulation,compaction molding granulation, cracking granulation, spray granulation,and injection granulation, coating methods such as pan coating, fluidlayer coating, and dry coating, puff dry, excess steam method, foam matmethod, expansion methods such as microwave incubation method, andextrusion methods with extrusion granulation machines and extruders.

The food, drink or feed of the present invention includes any food,drink or feed which comprises the binder microorganism of the inventionor fragment thereof as active ingredient. The active ingredient in thefood, drink or feed is not specifically limited to any concentration aslong as the resulting food, drink or feed can exert its activity ofspecifically binding to mutans Streptococci. The concentration of theactive ingredient is preferably 0.001 to 100% by weight, more preferably0.01 to 100% by weight and most preferably 0.1 to 100% by weight of thefood, drink or feed comprising such active ingredient or with respect tothe cell number those described herein.

Specific foods or drinks, to which the active ingredient is added,include, for example, juices, refreshing drinks, soups, teas, sour milkbeverages, dairy products such as fermented milks, ices, butter, cheese,processed milk and skim milk, meat products such as ham, sausage, andhamburger, fish meat cake products, egg products such as seasoned eggrolls and egg curd, confectioneries such as cookie, jelly, snacks, andchewing gum, breads, noodles, pickles, smoked products, dried fishes andseasonings. The form of the food or drink includes, for example, powderfoods, sheet-like foods, bottled foods, canned foods, retort foods,capsule foods, tablet foods and fluid foods.

The food or drink according to the invention to be ingested by infants,i.e. comprising the binder microorganism of fragment thereof with anactivity to specifically bind to mutans Streptococci, is preferably anutritious composition for infants. Such nutritious composition forinfants includes modified milk prepared for infants, protein-decomposedmilk, specific nutritionally modified milk or baby foods and foodsprepared for toddlers. The form of the nutritious composition forinfants includes but is not specifically limited to powder milks driedand pulverized and baby foods and also include general foods such as icecream, fermented milk, and jelly for infantile ingestion.

The nutritious composition for infants, and also the nutritious food orfeed composition for animals and particularly pet animals, in accordancewith the present invention is principally composed of protein, lipid,saccharide, vitamins and/or minerals. In the nutritious composition, theactive ingredient is blended with these components. The protein includesmilk proteins such as skim milk, casein, cheese whey, whey proteinconcentrate and whey protein isolates and their fractions such as alphas-casein, beta-casein, alpha-lactoalbumin and beta-lactoglobulin.Further, egg protein such as egg yolk protein, egg white protein, andovalbumin, or soybean protein such as defatted soybean protein,separated soybean protein, and concentrated soybean protein can be used.Other than these, proteins such as wheat gluten, fish meat protein,cattle meat protein and collagen may also be used satisfactorily.Further, fractions of these proteins, peptides from the acid or enzymetreatment thereof, or free amino acids maybe used satisfactorily aswell. The free amino acids can serve as nitrogen sources and canadditionally be used to give specific physiological actions. Such freeamino acids include, for example, taurine, arginine, cysteine, cystineand glutamine. For dogs, these include arginine, methionine, histidine,phenylalanine, isoleucine, threonine, leucine, tryptophan, lysine andvaline. For cats, taurine is also essential. The lipid includes animalfats and oils such as milk fat, lard, beef fat and fish oil, vegetableoils such as soybean oil, rapeseed oil, corn oil, coconut oil, palm oil,palm kernel oil, safflower oil, perilla oil, linseed oil, eveningprimrose oil, medium chain fatty acid triglyceride, and cotton seed oil,bacterially generated fats and oils, and fractionated oils thereof,hydrogenated oils thereof, and ester exchange oils thereof. The amountof lipid to be blended varies depending on the use.

Further preferred ingredients of food and feed, particularly pet feed,are omega-6 fatty acids and omega-3 fatty acids. Particularly preferredare linoleic acid, preferably in the form of corn, soy, canola,safflower and sunflower oil, whole grains and/or body fat of poultry;arachidonic acid, preferably in the form of body fat of poultry, leanmeat, egg yolks and/or fish oil; gamma linolenic acid, preferably in theform of black currant seed oil, borage oil and/or evening primrose oil;dihomogamma linolenic acid, preferably in the form of spleen, kidney,adrenals and/or metabolized from gla; alpha linolenic acid, preferablyin the form of flaxseed oil, canola, soy, and/or walnut oils;eicosapentaenoic acid, preferably in the form of cold water fish andtheir oil; docosahexaenoic acid, preferably in the form of cold waterfish and their oil.

The saccharide includes, for example, one or more of starch, solublepolysaccharides, dextrin, monosaccharides such as sucrose, lactose asdescribed herein, maltose, glucose, and fructose and otheroligosaccharides. Preferred saccharides include glucose, fructose,honey, galactose, lactose, sucrose, maltose, dextrins, glycogen andstarch.

However, as described above the maximum content of glucose, fructose,honey, galactose, lactose, sucrose and maltose preferably is 20 wt.-% ofthe total composition, more preferably 5 wt.-% of the total composition,and most preferably 2 wt.-% of the total composition. Even morepreferably, the aforementioned maximum content of 20 wt.-%, morepreferably 5 wt.-% and most preferably 2 wt.-% applies to the total ofall substances of the group of cane sugar, caramel, corn molasses, cornsyrup, dextrose, fructose, galactose, glucose, honey, lactose, levulose,maltose, mannitol, saccharin, sorbitol, sucrose and xylitol, and mostpreferably to the total of mono- and disaccharides.

Also preferred are dietary fiber preferably selected form cellulose,hemicellulose, pectin, plant gums and mucilages, beet pulp, guar gum,gum arabic, xanthan gum and locust bean gum. The total amount of suchsaccharide is preferably 40 to 80% by weight to the total solid in thenutritious composition. Further, artificial sweeteners such as aspartamemay be used satisfactorily. The amount of an artificial sweetener isappropriately 0.05 to 1.0% by weight per the total solid in thenutritious composition.

The vitamins include, but are not limited to, lycopene as an essentialcomponent and additionally include, for example, vitamins such asvitamin A, vitamin B group, vitamins C, D, and E and vitamin K group,folic acid, pantothenic acid, nicotinamide, carnitine, choline, inositoland biotin as long as such vitamins can be administered to infants orpet animals. Such vitamins are preferably from 10 mg to 5 g by weightper the total solid in the nutritious composition for infants. Preferredvitamins for nutritious compositions for infants and/or animals,preferably pet animals, include:

Vitamin A (Retinol), and/or beta carotene as precursor. The vitamin ispreferably present in the form of liver, fish liver oil, carrots, greenleafy vegetables, egg yolks and/or yellow fruits or in form of syntheticforms of Vitamin A and/or beta-carotene.

Vitamin D (Calciferol). The vitamin is preferably present in the form ofhalibut and/or cod liver oil, saltwater fish, cheese, yogurt and/or eggsor in form of synthetic forms of Vitamin D.

Vitamin E (Tocopherol). The vitamin is preferably present in the form ofgerm, corn, nuts, seeds, spinach and/or other green leafy vegetables,asparagus, vegetable oils or in form of synthetic forms of Vitamin E.

Vitamin K (Naphthoquinone). The vitamin is preferably present in theform of cabbage, cauliflower, spinach and/or other green leafyvegetables, cereals, soybeans, and/or other vegetables or in form ofsynthetic forms of Vitamin K. Preferably, the food or feed does notcomprise menadione.

Vitamin B1 (Thiamine). The vitamin is preferably present in the form ofwheat germ, rice and/or other whole grains, lean meats (especiallypork), liver, fish, yeast, dried beans, peas and/or soybeans or in formof synthetic forms of Vitamin B1.

Vitamin B2 (Riboflavin). The vitamin is preferably present in the formof lean meats, liver, fish, eggs, yeast, cheese, legumes, nuts and/orgreen leafy vegetables or in form of synthetic forms of Vitamin B2.

Vitamin B3 (Niacin). The vitamin is preferably present in the form ofliver, lean meat, poultry, fish, nuts, yeast, legumes, asparagus, seedsand/or green leafy vegetables or in form of synthetic forms of VitaminB3.

Vitamin B5 (Pantothenic Acid). The vitamin is preferably present in theform of eggs, fish, lean beef, legumes, yeast, broccoli and/or othervegetables in the cabbage family, white and/or sweet potatoes or in formof synthetic forms of Vitamin B5.

Vitamin B6 (Pyridoxine). The vitamin is preferably present in the formof meat, fish, eggs, bananas and/or whole grains or in form of syntheticforms of Vitamin B6.

Vitamin B8 (Biotin). The vitamin is preferably present in the form ofraw egg yolk, liver and/or vegetables or in form of synthetic forms ofVitamin B8.

Vitamin B9 (Folic Acid, Folate). The vitamin is preferably present inthe form of carrots, yeast, liver, egg yolks, melon, apricots, pumpkin,beans, rye, whole wheat and/or green leafy vegetables or in form ofsynthetic forms of Vitamin B9.

Vitamin B12 (Cobalamin). The vitamin is preferably present in the formof fish, liver, meat, poultry, eggs and/or cheese or in form ofsynthetic forms of Vitamin B12.

Vitamin C (Ascorbic Acid). The vitamin is preferably present in the formof citrus fruit juice or pulp, berries, tomatoes, cauliflower, potatoes,green leafy vegetables and/or green peppers or in form of syntheticforms of Vitamin C. Supplementation in an appropriate form, preferablyas calcium ascorbate, is preferred due to its beneficial effects on dogssuffering from chronic joint and musculoskeletal disorders. In puppiesit helps to prevent the development of such disorders.

Further, the minerals include calcium, magnesium, potassium, sodium,iron, copper, zinc, phosphorus, chlorine, manganese, selenium andiodine. Such minerals are preferably from 1 mg to 5 g by weight per thetotal solid in the nutritious composition for infants.

Other than those components described above, the nutritious compositionfor infants, and also the food or feed composition for animals,preferably pet animals, as used in accordance with the present inventionmay be blended with any component desirably blended in nutritiouscompositions, for example, dietary fiber, nucleotides, nucleic acids,flavors, and colorants.

The food or drink as used in accordance with the present invention canbe used as a health food or drink or a functional food or drink toprevent and/or treat caries and/or to prevent or treat oral malodourand/or to prevent or treat dental calculus.

When the food or drink according to the present invention is ingested,the amount to be ingested is not specifically limited. The amount to beingested is generally 0.1 to 50 g, preferably 0.5 g to 20 g daily, basedon the total amount of active ingredient.

The food or drink is continuously ingested at this amount for a periodfrom a single day up to 5 years, preferably from 2 weeks to one year.Herein, the amount ingested can be adjusted to an appropriate rangedepending on the severity of the symptom of the individual ingesting thefood or drink, the age and body weight thereof, and the like.

The composition of the present invention may comprise, further to thebinder microorganism or fragment thereof, cereals, brans, oil-seedmeals, animal-derived raw feed materials, other raw feed materials andpurified products. The cereals can include, mile, wheat, barley, oats,rye, brown rice, buckwheat, fox-tail millet, Chinese millet, Deccangrass, corn, and soybean. The brans can include, rice bran, defattedrice bran, bran, lowest-grade flour, wheat germ, barley bran, screeningpellet, corn bran, and corn germ. The oil-seed meals include, forexample, soybean meal, soybean powder, linseed meal, cottonseed meal,peanut meal, safflower meal, coconut meal, palm meal, sesame meal,sunflower meal, rapeseed meal, kapok seed meal and mustard meal. Theanimal-derived raw feed materials include, for example, fish powders,import meal, whole meal, and coast meal, fish soluble, meat powder, meatand bone powder, blood powder, decomposed hair, bone powder, byproductsfrom butchery, feather meal, silkworm pupa, skim milk, casein, dry wheyand krill. Other raw feed materials include, for example, plant stemsand leaves such as alfalfa, hey cube, alfalfa leaf meal, and locust leafpowder, byproducts from corn processing industries, such as corn glutenmeal, corn gluten feed and corn steep liquor, starch, sugar, yeast,byproducts from fermentation industry such as beer residue, malt root,liquor residue and soy sauce residue, and agricultural byproducts suchas citrus processed residue, soybean curd residue, coffee residue, andcocoa residue, cassava, horse bean, guar meal, seaweed, spirulina andchlorella. The purified products include, for example, proteins such ascasein and albumin, amino acids, starch, cellulose, saccharides such assucrose and glucose, minerals and vitamins.

The composition of the present invention may further comprise one ormore additives. Such additive for foods can be produced by a generalmethod for producing additives for foods, drinks or feeds. If necessary,additives for general use in foods, drinks or feeds, for example,additives described in Food Additive Handbook (The Japan Food AdditivesAssociation; issued on Jan. 6, 1997) may be added satisfactorily,including sweeteners, colorants, preservatives, thickeners andstabilizers, antioxidants, color fixing agents, bleaches, antiseptics,gum base, bitters, enzymes, brightening agents, acidifier, seasonings,emulsifiers, enhancers, agents for manufacture, flavors, and spiceextracts. Further, conventional saccharides, starch, inorganicmaterials, plant powders, excipients, disintegrators, lubricants,binders, surfactants, and plasticizers mentioned previously forpharmaceutical tablets may be added satisfactorily.

The sweeteners include Brazzein; Curculin; Erythritol; Glycyrrhizin;Glycerol, E422; Hydrogenated starch hydrolysates; Inulin; Isomalt, E953;Lactitol, E966; Luo han guo; Mabinlin; Maltitol, E965;Malto-oligosaccharide; Mannitol, E421; Miraculin; Monatin; Monellin;Pentadin; Sorbitol, E420; Stevia extracts or steviol glycosides,particularly Stevioside, Rebaudioside A, Rebaudioside C, Dulcoside A,Rubusoside, Steviolbioside H and Rebaudioside B; Tagatose; Thaumatin,E957; Xylitol, E967; Acesulfame potassium, E950; Alitame; Aspartame,E951; Salt of aspartame-acesulfame, E962; Cyclamate, E952; Dulcin;Glucin; Neohesperidin dihydrochalcone, E959; Neotame; P-4000; Saccharin,E954; Sucralose, E955; licorice; xylose and rakanka (Momordicagrosvenori fruit).

The colorants include carotenoid and turmeric oleoresin, flavonold,caramel color, spirulina color, chlorophyll, purple sweet potato color,purple yam color, perilla color, and blueberry color.

The preservatives include, for example, sodium sulfite, benzoates,benzoin extract, sorbates, and propionates.

The thickeners and stabilizers include, for example, gums such as gumarable and xanthan gum, alginates, chitin, chitosan, aloe extract, guargum, hydroxypropyl cellulose, sodium casein, corn starch, carboxymethylcellulose, gelatin, agar, dextrin, methyl cellulose, polyvinyl alcohol,microfiber cellulose, microcrystalline cellulose, seaweed cellulose,sodium polyacrylate, sodium polyphosphate, carrageenan or yeast cellwall.

The anti-oxidants include, for example, vitamin C group, sodiumethylenediaminetetraacetate, calcium ethylenediaminetetraacetate,erythorbic acid, oryzanol, catechin, quercetin, clove extract,enzyme-treated rutin, apple extract, sesame seed extract,dibutylhydroxytoluene, fennel extract, horseradish extract, water celeryextract, tea extract, tocopherols, rapeseed extract, coffee beanextract, sunflower seed extract, ferulio acid, butylhydroxyanisole,blueberry leaf extract, propolis extract, pepper extract, garden balsamextract, gallic acid, eucalyptus extract, and rosemary extract.

The color fixing agents include, for example, sodium nitrite. Thebleaches include, for example, sodium sulfite.

The antiseptics include, for example, o-phenyl phenol. The gum baseincludes, for example, acetylricinoleate methyl, urushi wax, ester gum,elemi resin, urucury wax, kaurigum, carnaubawax, glycerin fatty acidester, spermaceti wax, copaibabalsam, copal resin, rubber, rice branwax, cane wax, shellac, jelutong, sucrose fatty acid ester,depolymerized natural rubber, paraffin wax, fir balsam, propylene glycolfatty acid ester, powdered pulp, powdered rice hulls, jojoba oil,polyisobutylene, polybutene, microcrystalline wax, mastic gum, bees waxand calcium phosphate. The bitters include, for example, isoalpha-bitteracid, caffeine, kawaratake (Coriolus versieolor) extract, redbarkcinchona extract, Phellodendron bark extract, gentian root extract,spice extracts, enzymatically modified naringin, Jamaica cassia extract,theabromine, naringin, cassia extract, absinth extract, isodonisextract, olive tea, bitter orange (Citrus aurantium) extract, hopextract and wormwood extract. The enzymes include, for example, amylase,trypsin or rennet. The brightening agents include, for example, urushiwax and japan wax. The acidifier include, for example, adipic acid,itacania acid, citric acids, succinic acids, sodium acetate, tartaricacids, carbon dioxide, lactic acid, phytic acid, fumario acid, malicacid and phosphoric acid. The seasonings include, for example, aminoacids such as asparagine, aspartic acid, glutamic acid, glutamine,alanine, isoleucine, glycine, serine, cystine, tyrosine, leucine, andpraline, nucleic acids such as sodium inosinate, sodium uridinate,sodium guanylate, sodium cytidylate, calcium ribonucleotide and sodiumribonucleotide, organic acids such as citric acid and succinic acid,potassium chloride, sodium chloridedecreased brine, crude potassiumchloride, whey salt, thpotassium phosphate, dipotassium hydrogenphosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate,sodium dihydrogen phosphate, trisodium phosphate and chlorella extract.

The enhancers include, for example, zinc salts, vitamin C group, variousamino acids, 5-adenylic acid, iron chloride, hesperidin, variouscalcined calcium, various non-calcined calcium, dibenzoylthiamine,calcium hydroxide, calcium carbonate, thiamine′ hydrochloride salt,Dunallella. Oarotene, tocopherol, nicotinic acid, carrot carotene, palmoil carotene, calcium pantothenate, vitamin A, hydroxyproline, calciumdihydrogen pyrophosphate, ferrous pyrophosphate, ferric pyrophosphate,ferritin, heme iron, menaquinone, folic acid and riboflavine.

The agents for manufacture include, for example, processing auxiliariessuch as acetone and ion exchange resin.

The flavors include, for example, vanilla essence and the spice extractsinclude, for example, capsicum extract.

These various additives can be added to the active ingredient, takinginto consideration the mode of administration, in accordance with thepresent invention.

It is envisaged that the composition of the present invention,preferably the food or feed composition or pharmaceutical composition,comprises the above mentioned binder microorganism belonging to thegroup of lactic acid bacteria in the form of a probiotic microorganism.Namely, in addition to the probiotic effect, the above mentionedprobiotic microorganism belonging to the group of lactic acid bacteriais useful for treating and/or preventing biofilm formation caused bymutans Streptococci. The amount of said probiotic microorganism is highenough to significantly positively modify the condition to be treated,preferably caries, dental calculus and/or oral malodour, but low enoughto avoid serious side effects (at a reasonable benefit/risk ratio),within the scope of sound medical judgment. An effective amount of saidprobiotic microorganism will vary with the particular goal to beachieved, the age and physical condition of the patient being treated,the severity of the underlying disease, the duration of treatment, thenature of concurrent therapy and the specific microorganism employed.The effective amount of said probiotic microorganism will thus be theminimum amount which will provide the desired specific binding to mutansStreptococci. The presence of, for example, 1×10⁹ bacteria, as viable ornon-viable whole cells, in 0.05 ml solution of phosphate buffered salinesolution, or in 0.05 ml of suspension of agar, or the dry weightequivalent of cell wall fragments, is effective when administered inquantities of from about 0.05 ml to about 20 ml. A decided practicaladvantage is that the probiotic organism may be administered in aconvenient manner such as by the oral route. Depending on the route ofadministration, the active ingredients which comprise said probioticorganisms may be required to be coated in a material to protect saidorganisms from the action of enzymes, acids and other natural conditionswhich may inactivate said organisms. In order to administer probioticorganisms by other than parenteral administration, they should be coatedby, or administered with, a material to prevent inactivation. Forexample, probiotic organisms may be co-administered with enzymeinhibitors or in liposomes. Enzyme inhibitors include pancreatic trypsininhibitor, diisopropylfluorophosphate (DFP) and trasylol. Liposomesinclude water-in-oil-in-water P40 emulsions as well as conventional andspecifically designed liposomes which transport lactobacilli or theirby-products to the urogenital surface. Dispersions can also be prepared,for example, in glycerol, liquid polyethylene glycols, and mixturesthereof, and in oils. Generally, dispersions are prepared byincorporating the various sterilized probiotic organisms into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof. Additional preferred methods of preparation include but are notlimited to lyophilization and heat-drying.

The composition of the present invention also encompasses productsintended to be administered orally, or buccal, which comprise anacceptable pharmaceutical carrier as described herein to which, or ontowhich, cells of the above mentioned microorganism belonging to the groupof lactic acid bacteria is added in fresh, concentrated or dried form,for example. These products may be provided in the form of an ingestiblesuspension, a gel, a diffuser, a capsule, a hard gelatin capsule, asyrup, or in any other galenic form known to persons skilled in the art.

When the probiotic organisms are suitably protected as described above,the active compound may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or it may beenclosed in hard or soft shell gelatin capsule, or it may be compressedinto tablets designed to pass through the stomach (i.e., entericcoated), or it may be incorporated directly with the food of the diet.For oral therapeutic administration, the probiotic organisms may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Compositions or preparations according to the presentinvention are prepared so that an oral dosage unit form contains, forexample, about 1×10⁹ viable or non-viable cells per ml, e.g.lactobacilli per ml. The probiotic organism is compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically or food acceptable carrier in dosage unit form ashereinbefore disclosed. A unit dosage form can, for example, contain theprincipal active compound in an amount approximating 10⁹ viable ornon-viable, e.g., lactobacilli, per ml. In the case of compositionscontaining supplementary ingredients such as prebiotics, the dosages aredetermined by reference to the usual dose and manner of administrationof the said ingredients.

Preferred ingredients in the food and feed composition according to thepresent invention are alfalfa, alfalfa concentrate powder, alfalfadehydrated meal, alfalfa nutrient concentrate, alpha-lipoic acid, animaldigest, animal fat, argenine, artificial flavor, ascorbic acid,asparagus, bacillus subtilis, bacon, bacon flavors, barley, barleygrass, barley malt flour, basil, beans, beef, beef & bone meal, beefbroth, beef by-products, beef flavor, beef liver, beef meal, beeftallow, beet pulp, beet pulp (sugar removed), beets, beta carotene, BHA,bifidobacterium longum, bifidobacterium pseudolongum, bifidobacteriumthermophilum, biotin, blue 2, blue 2 and other color, blueberries, bonemeal, borage oil, brewers dried yeast, brewers yeast, brewers yeastextract (saccharomyces cerevisiae fermentation solubles), brewer's rice,broccoli, brown rice, brown rice flour, calcium, calcium ascorbate,calcium carbonate, calcium chloride, calcium iodate, calciumpantothenate, calcium phosphate, calcium propionate, calcium sulfate,cane molasses, canola meal, canola oil, canthaxanthin, caramel, caramelcolor, carmine, carrageenan, carrageenan gum, carrot powder, carrots,casein, catfish, catfish meal, celery, cellulose, cellulose powder,cheese powder, chelated cobalt, chelated copper, chelated iron, chelatedpotassium, chicken, chicken broth, chicken by products organs only,Chicken by-product, chicken by-product meal, chicken by-products organmeat only, chicken cartilage natural, chicken fat, chicken fatnaturally, chicken flavors, chicken giblets, chicken liver, chickenliver digest, chicken liver flavor, chicken meal, chicken natural,chicken stock, chicory extract, choline chloride, chondroitine sulfate,cinnamon, citric acid, citric acid and rosemary, citric acid androsemary extract, citrus pectin, clove bud oil, cobalt amino acidchelate, cobalt carbonate, cobalt proteinate, cod, copper, copper aminoacid chelate, copper amino acid complex, copper oxide, copperproteinate, copper sulfate, corn, corn bran, corn flour, corn germ meal,corn gluten, corn gluten meal, corn grits, corn meal, corn oil, cornstarch, corn starch-modified, corn syrup, cracked barley, crackedpearled barley, cranberries, cranberry powder, deboned chicken, debonedlamb, deboned turkey, dehydrated alfalfa, dehydrated alfalfa meal,dehydrated carrots, dehydrated potatoes, dextrose, DHA, dicalciumphosphate, DL-alpha tocopherol acetate, DL-methionine, dried animaldigest, dried apples, dried bacillus licheniformis fermentation extract,dried bacillus subtilis fermentation extract, dried beet pulp, driedbeet pulp (sugar removed), dried blueberries, dried brewers yeast, driedbuttermilk, dried capsicum, dried carrots, dried cellulose, driedcheddar cheese, dried cheese, dried cheese powder, dried chickencartilage, dried chicken liver, dried chicken stock, dried chickoryroot, dried citrus pulp, dried cooked turkey, dried cranberries, driedegg, dried egg powder, dried egg product, dried eggs, dried garlic,dried ginger, dried grape pomace, dried green beans, dried kelp, driedkelp meal, dried liver digest, dried meat by-product, dried paprika,dried parsley flakes, dried peas, dried plain beet pulp, dried potatoes,dried spinach, dried sweet potato, dried tomato pomace, dried vegetablefiber carrots, dried whey, dried yam, duck, durum flour, durum semolinaenriched with thiamine mononitrate, egg noodles, egg pieces, eggproduct, eggs, enterococcus faecium, ethoxyquin, eucalyptus oil, ferroussulfate, fiber, fish, fish broth, fish meal, fish meal natural, fishoil, flax meal, flax seed, flaxseed meal, folic acid, folic acidpyridoxine hydrochloride, food starch, fresh, fresh chicken, freshchicken by-products, fructooligosaccharides, fumaric acid, garlic,garlic extract, garlic flavor, garlic oil, garlic powder, gelatin,ginger, ginger extract, glucosamine, glucosamine hydrochloride,glycerin, glycerine, glyceryl monostearate, glycine, green beans, greentea, ground corn, ground flax seed, ground psyllium seed, ground rice,ground wheat, ground wheat flour, ground whole grain barley, groundwhole grain corn, ground whole grain sorghum, ground whole grain wheat,ground whole peas, ground whole wheat, ground yellow corn, guar gum, gumarabic, halibut, herring meal, herring oil, hydrochloric acid, inositol,iodine, iodized salt, iron amino acid chelate, iron amino acid complex,iron oxide, iron proteinate, iron sulfate, L-alanine, L-arginine,L-ascorbyl-2-polyphosphate, L-ascorbyl-2-polyphosphate a, L-carnitine,L-lysine, L-lysine monohydrochloride, L-tryptophan, lactobacillusacidophilus, lamb, lamb broth, lamb by-product, lamb digest, lamb fat,lamb liver, lamb meal, lamb stock, lamb tripe, lecithin, lentils,lettuce, liver, locust bean gum, lutein, lycopene, lysine, mackerel,magnesium oxide, malt extract, malted barley flour, managanous sulfate,manganese amino acid chelate, manganese oxide, manganese proteinate,manganese sulfate, manganous oxide, manganous oxide calcium iodate,manganous proteinate, manganous sulfate, maple syrup, marigold extract,marigold meal, meat and bone meal natural, meat and liver meal, meatby-products, menadione dimethylpyrimidinol bisulfite, menadione sodiumbisulfite complex, menadione vitamin K3, menhaden fish meal, menhadenfish oil, milk, mixed tocopherols, mixed vegetable fiber carrots,modified food starch, modified starch, molasses, monocalcium phosphate,monosodium phosphate, natural and artificial chicken flavor, natural andartificial flavors, natural chicken flavor, natural color, naturalflavor, natural poultry flavor, natural smoke flavor, niacin, niacin &ferrous sulfate, non-fat yogurt, oat bran, oat fiber, oat groats, oatmeal, oats, ocean fish, ocean fish meal, ocean whitefish, omega fattyacids, onion extract, onion powder, pantothenate, paprika oleoresin,parsley, parsley flakes, parsley oil, parsley powder, partiallyhydrogenated soybean oil, pasta, pea fiber, pea protein, peanut hulls10.8%, pearled barley, peas, peppers, petrolatum, philloquinone vitaminK1, phosphoric acid, pork broth, pork by-products, pork liver,postassium sorbate, potassium amino acid complex, potassium chloride,potassium citrate, potassium iodide, potassium iodine, potassiumsorbate, potato, potato fiber, potato starch, poultry, poultryby-product meal, poultry by-products, poultry fat, poultry giblets,poultry liver, powdered cellulose, powdered cellulose 11.1%, propionicacid, propyl gallate and citric acid, propylene glycol, pyridoxinehydrochloride, pysllium, rabbit, rabbit by-products, rabbit stock, red3, red 40 and other color, red peppers, riboflavin, rice, rice bran,rice flour, rice gluten, rice hulls, rice protein concentrate, rosemary,rosemary extract, rosemary extract and citric acid, rye, sage, salmon,salmon broth, salmon meal, salmon oil, salt, sea salt, selenium, shrimp,smoke flavor, sodium alginate, sodium ascorbate, sodium bisulfate,sodium carbonate, sodium caseinate, sodium chloride, sodiumhexametaphosphate, sodium metabisulfate, sodium nitrite for colorretention, sodium phosphate, sodium propionate, sodium selenite, sodiumsilico aluminate, sodium tripolyphoshate, sodium tripolyphosphate,sorbic acid, sorbitol, soy flour, soy hulls, soy lecithin, soy proteinconcentrate, soy protein isolate, soya oil, soybean hulls, soybean meal,soybean mill run, soybean oil, spearmint, spinach, spirulina, starch,steamed bone meal, sucrose, sufficient water for processing, sugar,sun-cured alfafa meal, sunflower meal, sunflower oil, sweet potatopowder, sweet potatoes, tallow, tapioca starch, taurine, tetra sodiumpyrophosphate, textured vegetable protein, thiamine, thiaminehydrochloride, thiamine mononitrate, thyme, titanium dioxide, titaniumdioxide color, tomato flakes, tomato paste, tomato pomace, tomatos,trace minerals (calcium sulfate), trace minerals (copper sulfate), traceminerals (potassium chloride), trace minerals (sodium tripolyphoshate),trace minerals (zinc oxide), trace minerals (zinc proteinate), traceminerals (zinc sulfate), tricalcium phosphate, tuna, tuna meal, turkey,turkey broth, turkey by-product meal, turkey natural, turkey stock,turmeric, veal, veal broth, vegetable oil, venison, venison by-products,venison liver, venison meal, venison meat, venison stock, vitamin A,vitamin A & D3, vitamin A acetate, vitamin B1, vitamin B12, vitamin B12and D3, vitamin B2, vitamin B6, vitamin C, vitamin D3, vitamin D3 and E,vitamin E, vitamin K, water, water cress, water sufficient forprocessing, watercress and spinach, wheat, wheat bran, wheat flour,wheat germ meal, wheat gluten, wheat middlings, wheat mill run, wheatstarch, whey, white fish, whitefish, whitefish meal, whole brown rice,whole carrots, whole cranberries, whole eggs, whole garlic cloves, wholegrain corn, whole grain wheat, whole ground barley, whole ground brownrice, whole ground oats, whole ground wheat, whole rice, whole sweetpotatoes, whole wheat, whole wheat flour, wild rice, xanthan gum, yeastculture, yellow 5, yellow 6, yellow squash, yellow zucchini, yuccaschidigera, yucca schidigera extract, zinc amino acid chelate, zincamino acid complex, zinc oxide, zinc proteinate, zinc proteinate andzinc sulfate.

Most preferred ingredients are: alpha-lipoic acid, preferably in anamount sufficient to promote formation of healthy skin and/or coat; forantibiotic purposes preferred ingredients are alpha-lipoic acid, driedgarlic, garlic extract, garlic oil and garlic powder; lecithin,preferably as an antioxidans; cracked barley, cracked pearled barley,ground whole grain corn, ground whole grain sorghum, herring oil,mackerel, menhaden fish oil, ocean fish, whole brown rice and wholesweet potatoes are preferred as sources of energy; preferred sources offiber are dehydrated carrots, dried apples, ground whole grain barley,oat bran, oat groats, oat meal, whole ground barley, whole ground oats;cobalt proteinate (source of chelated cobalt), copper proteinate, ironproteinate, manganese proteinate, manganous proteinate, trace mineralsand, zinc proteinate are preferred sources of minerals; preferredsources of omega-3 fatty acids are borage oil, canola oil, canola oil(preserved with mixed tocopherols) and flax seed; rosemary extract andcitric acid are preferred preservatives; preferred sources of proteinare beef meal, chicken meal, dried peas, halibut, lamb meal, menhadenfish meal, ocean whitefish, peas, venison meal, whole ground brown riceand whole ground wheat; preferred sources of vitamins are calciumascorbate, carrots, dried carrots, mixed tocopherols, whole carrots;preferred replacements for plain water, and also preferred flavours, arebeef broth, chicken broth, lamb broth, lamb stock and turkey broth.

Ingredients not most but also highly preferred are: alfalfa concentratepowder, alfalfa dehydrated meal, chicken fat, chicken liver, fresh,ocean fish meal and pearled barley as sources of energy; green beans,ground whole peas, oats and pea fiber as sources of fiber; cobalt aminoacid chelate, copper amino acid chelate, copper amino acid complex,dried kelp meal, iron amino acid chelate, manganese amino acid chelate,manganese sulfate, potassium amino acid complex, zinc amino acid chelateas sources of minerals; chicory extract, ginger extract and yuccaschidigera extract as prebiotics; good preservatives are garlic,rosemary and sage; alfalfa nutrient concentrate, catfish, catfish meal,cod, duck, eggs, ground whole wheat, herring meal, pea protein, rabbit,shrimp, tuna, venison, venison meat, white fish, whitefish, whitefishmeal, whole wheat, are preferred as sources of protein; beta caroteneand folic acid are preferred sources of vitamins; chicken stock, rabbitstock, turkey stock, veal broth and venison stock are preferredreplacements for plain water, and also preferred flavours.

A standard food of feed composition of the present invention, preferablya pet food, preferably comprises one or more ingredients of the groupconsisting of vitamin B-12, biotin, calcium carbonate, calciumpantothenate, choline chloride, cobalt carbonate, copper sulfate,vitamin D3 and E supplements, DL-methionine, dried kelp, ferroussulfate, folic acid, inositol, manganese oxide, manganous oxide,manganous sulfate, menadione sodium bisulfite complex, mineralsupplements including zinc sulfate, natural flavor, niacin and ferroussulfate, niacin supplement, potassium chloride, potassium iodide,pyridoxine hydrochloride, riboflavin, sodium selenite, taurine,thiamine, thiamine mononitrate, thiamine mononitrate, vitamin A, vitaminA & D3, vitamin A acetate, vitamin B12, vitamin D3, water, zinc oxideand zinc sulfate.

The feed as used in accordance with the present invention maybe any feedcomprising the binder microorganism or fragment thereof as an activeingredient. The feed includes, for example, pet feeds for dogs, cats andrats, and cattle feeds for cows and pigs.

The feed can be produced by appropriately blending the active ingredientas described herein above in a raw feed material including, for example,cereals, brans, oil-seed meals, animal-derived raw feed materials, otherraw feed materials and purified products as indicated above. Preferredfeeds of the present invention are chew products. Preferred products arepig ears, bull sinews, cattle tails, oesophagus, dried muscle meat, pigfeet and dried pressed cowhide, buffalo hide or composite chew products,containing plant materials as e.g. fibers, brans etc. Other chewproducts are known in the art and are also preferred, for example thechew products of U.S. Pat. No. 2,988,045, WO 01/50882 A2, EP 1 151 674A1, EP 1 006 789 A1 and CH 676200 A5. The chew product preferably is inthe shape of a bone, a roll, a donut, a bow, a pretzel, a figure eight,or a chip.

In case of providing to animals the feed according to the presentinvention, the amount of the feed to be ingested is not specificallylimited but is preferably, for example, 0.1 mg to 50 g per 1 kg bodyweight per day, preferably 0.5 mg to 20 g per 1 kg body weight per day,based on the amount of the active ingredient. The feed is continuouslyingested at this amount for a period from a single day up to 5 years,preferably from 2 weeks to one year. Again, the amount ingested can beadjusted to an appropriate range depending on the species, age and bodyweight of the animal ingesting the feed, and the like.

It is likewise preferred that the composition of the present inventioncomprises one or more probiotic microorganisms or products obtainedthereof, independently of the binder microorganism and fragment thereof.Preferred probiotic microorganisms and respective products thereof arebacillus subtilis, bifidobacterium longum, bifidobacterium pseudolongum,bifidobacterium thermophilum, dried bacillus licheniformis fermentationextract, dried bacillus subtilis fermentation extract, enterococcusfaecium and lactobacillus acidophilus.

Particularly preferred are food and feed compositions furthercomprising, in addition to the binder microorganism or fragment thereof,a further microorganism for preventing and/or treating oral malodor.Most preferred are food and feed compositions further comprising suchmicroorganism as disclosed in WO 2009/149816 A1, which is incorporatedherein in its entirety, Of these, the further microorganism ispreferably selected from the group consisting of Lactobacillusacidophilus having DSMZ accession number DSMZ 19825, DSMZ 19826, DSMZ19827.

The invention is now further described by selected examples andembodiments. These embodiments and examples are intended to representcertain preferred features of the present invention, without limitingthe scope of this description or the scope of the claims. It is to beunderstood that the skilled artisan can devise further working examplesand embodiments by his common general knowledge and the instructions andexplanations given in this description and the documents incorporatedherein by reference.

EXAMPLE 1 Storage and Growth of Binder Microorganisms

Storage and growth of strains can be performed according to ordinaryprocedures. According to this present example, strains are stored asfrozen stocks at −80° C. 1 ml of a culture is grown to stationary phase(OD600: 4-8) in MRS-Medium and mixed with 500 μl of a sterile 50%glycerine solution and frozen. Cultures of mutans Streptococci are grownin TSY-media to stationary phase (OD600 1-2) and are treated asmentioned above for frozen storage.

Cultivation of mutans Streptococci (S. mutans, S. sobrinus, S. ratti, S.cricetus, S. ferus or S. macacae) as well as cultivation of lactobacillican be done in 5 ml in closed Falcon tubes at 37° C. without shackingover night.

In particular, the strains used in the present application were storedas frozen stocks at −80° C. 1 ml of a culture grown to stationary phase(OD600/ml_(—)4-8) in MRS-broth was mixed with 500 μl of a sterile 50%glycerol solution and frozen.

In particular, cultures of mutans Streptococci were grown in TSY-brothto stationary phase (OD600/ml_(—)1-2) and treated as mentioned above.

Cultivation of mutans Streptococci (S. mutans (DSMZ 20523, serotype c;NCTC 10923, serotype e; NCTC 11060, serotype f), S. sobrinus DSMZ 20742,S. ratti DSMZ 20564, S. cricetus DSMZ 20562, S. ferus DSMZ 20646 or S.macacae DSMZ 20714) and cultivation of lactobacilli was done in 5 ml inclosed Falcon tubes at 37° C. without shacking over night. For thefluorescence assays as described in Example 5 S. mutans DSMZ 20523 wasused.

For an aggregation assay the lactobacilli were grown in MRS-medium. 5 mlMRS—medium were inoculated with 10 μl of the stock and incubated for 3days at 37° C. under aerobic conditions. The optical density of theculture at 600 nm (OD600) was measured. The culture was then diluted toan OD600 of 2 using PBS-buffer. The mutans Streptococci were grown in 7ml TSY-medium. 7 ml of TSY-medium were inoculated with 10 μl of thestock and incubated at 37° C. under anaerobic conditions.

MRS-Broth:

MRS-mixture (Difco, USA) 55 g/l, pH: 6.5

TSY-Broth: TSY-mixture (Difco, USA) 30 g/l

Yeast extract (Deutsche Hefewerke, Germany) 3 g/l

Buffer: PBS-Buffer: Na₂HPO₄ 2H₂O 1.5 g/l KH₂PO₄ 0.2 g/l NaCl 8.8 g/l

pH adjusted with HCl

EXAMPLE 2 Taxonomic Classification of Binder Strains and StreptococcusStrains

The taxonomic classification of the strains was done according to theircarbohydrate fermentation pattern. This was determined using the API 50CH (bioMerieux, France) system and analyzed using APILAB PLUS softwareversion 3.3.3 (bioMerieux, France).

EXAMPLE 3 Staining of Cells

After the lactobacilli and the mutans Streptococci were grown asdescribed in Example 1, the mutans Streptococci were stained using afluorescence strain. For this, the OD600 of the culture was measured.The culture was harvested by centrifugation at 3200×g for min. thepellet was resuspended in PBS-buffer. The amount of buffer wascalculated so that the resulting suspension had an OD600 of 4.2 ml ofthat suspension were mixed with 2 μl of a CFDA-SE solution (Invitrogen,USA) that was prepared according to the manufacturer's instructions.Staining of the cells was carried out by incubating the mixture for 2 hat 37° C. The stained cells were harvested by centrifugation at 3200×gfor 5 min. The cells were subsequently resuspended in 2 ml PBS-buffer.

EXAMPLE 4 Pelleting Aggregation Assay of Mutans Streptococci

For the assay, mixing of lactobacilli and mutans Streptococci was donein volumetric ratios of 3:1 to 60:1 (mutans Streptococci:lactobacilli),this corresponds to a ratio of colony forming units from 1:50 to 1:2.5.

An optical density measured at a wavelength of 600 nm in 1 ml meanspreferably for mutans Streptococci 3×10⁸ colony forming units and forlactobacilli preferably 7×10⁹ colony forming units. Mixing was done in 2ml volume in 15 ml Falcon tubes. The culture suspensions were dilutedwith PBS-buffer to obtain the volumetric ratios mentioned above whilekeeping the final volume at 2 ml. The mixture was vortexed for 15seconds. An aggregation is visible as an immediate turbidity of thesuspension. The tubes were left undisturbed for 20 min, after thatperiod of time the aggregates settle as a visible pellet whereasnon-aggregating mixtures stay in suspension. The formed aggregates wereseparated by centrifugation at 500×g for 30 seconds. Afterwards, theamount of aggregation was quantified by measuring the amount ofnon-aggregated cells that were left in the supernatant. Correspondingly,1 ml of the supernatant was carefully removed to measure the opticaldensity. The optical density was measured at 600 nm. The value aftersubtraction of the respective control experiment without lactobacillirepresents the amount of cells that have not been aggregated.

As a control, self-aggregation of the respective Lactobacillus strainand the mutans Streptococcus strains was always investigated byperforming the test with only the Lactobacillus or the mutansStreptococcus strain added to the tube. An aggregation of S. mutans byLactobacillus is shown in FIGS. 1 (left tube) and 2.

The lactobacilli strains as described herein above, in particular thosedeposited with the DSMZ, exhibited aggregation of all S. mutansserotypes without showing a self-aggregation behaviour.

EXAMPLE 5 Fluorescence Aggregation Assay of Mutans Streptococci

For the assay, suspension of the respective lactobacillus and therespective stained mutans Streptococcus (S. mutans DSMZ 20523 withLb-OB-K1 (DSMZ 16667), S. mutans DSMZ 20523 with Lb-OB-K2 (DSMZ 16668),S. mutans DSMZ 20523 with Lb-OBK3 (DSMZ 16669), S. mutans DSMZ 20523with Lb-OB-K4 (DSMZ 16670), S. mutans DSMZ 20523 with Lb-OB-K5 (DSMZ16671), S. mutans DSMZ 20523 with Lb-OB-K6 (DSMZ 16672), S. mutans DSMZ20523 with Lb-OB-K7 (DSMZ 16673); S. sobrinus DSMZ 20742 with Lb-OB-KI(DSMZ 16667), S. sobrinus DSMZ 20742 with Lb-OB-K2 (DSMZ 16668), S.sobrinus DSMZ 20742 with Lb-OB-K3 (DSMZ 16669), S. sobrinus DSMZ 20742with Lb-OB-K4 (DSMZ 16670), S. sobrinus DSMZ 20742 with Lb-OB-K5 (DSMZ16671), S. sobrinus DSMZ 20742 with Lb-OB-K6 (DSMZ 16672), S. sobrinusDSMZ 20742 with Lb-OB-K7 (DSMZ 16673); S. cricetus DSMZ 20562 withLb-OB-KI (DSMZ 16667), S. cricetus DSMZ 20562 with Lb-OB-K2 (DSMZ16668), S. cricetus DSMZ 20562 with Lb-OB-K3 (DSMZ 16669), S. cricetusDSMZ 20562 with Lb-OB-K4 (DSMZ 16670), S. cricetus DSMZ 20562 withLb-OB-K5 (DSMZ 16671), S. cricetus DSMZ 20562 with Lb-OB-K6 (DSMZ16672), S. cricetus DSMZ 20562 with Lb-OB-K7 (DSMZ 16673); S. ratti DSMZ20564 with Lb-OB-K1 (DSMZ 16667), S. ratti DSMZ 20564 with Lb-OB-K2(DSMZ 16668), S. ratti DSMZ 20564 with Lb-OB-K3 (DSMZ 16669), S. rattiDSMZ 20564 with Lb-OB-K4 (DSMZ 16670), S. ratti DSMZ 20564 with Lb-OB-K5(DSMZ 16671), S. ratti DSMZ 20564 with Lb-OB-K6 (DSMZ 16672), S. rattiDSMZ 20564 with Lb-OB-K7 (DSMZ 16673);

S. ferus DSMZ 20646 with Lb-OB-K1 (DSMZ 16667), S. ferus DSMZ 20646 withLb-OBK2 (DSMZ 16668), S. ferus DSMZ 20646 with Lb-OB-K3 (DSMZ 16669), S.ferus DSMZ 20646 with Lb-OB-K4 (DSMZ 16670), S. ferus DSMZ 20646 withLb-OB-K5 (DSMZ 16671), S. ferus DSMZ 20646 with Lb-OB-K6 (DSMZ 16672),S. ferus DSMZ 20646 with Lb-OB-K7 (DSMZ 16673); S. macacae DSMZ 20724with Lb-OB-K1 (DSMZ 16667), S. macacae DSMZ 20724 with Lb-OB-K2 (DSMZ16668), S. macacae DSMZ 20724 with Lb-OB-K3 (DSMZ 16669), S. macacaeDSMZ 20724 with Lb-OB-K4 (DSMZ 16670), S. macacae DSMZ 20724 withLbOB-K5 (DSMZ 16671), S. macacae DSMZ 20724 with Lb-OB-K6 (DSMZ 16672)and S. macacae DSMZ 20724 with Lb-OB-K7 (DSMZ 16673)) were mixed. 50 μlof the lactobacillus suspension were added to 50 μl of stained mutansStreptococci in a 96 well microtiter plate. The plate was vortexed atfull speed for 12 minutes. Afterwards the plate was centrifuged at 500×gfor 10 seconds. The supernatant was carefully removed and discarded. Thepellet was resuspended in 100 μl of PBS-buffer The fluorescence of thesuspension was measured in a microtiterplate fluorescence reader at awavelength of 495 nm for excitation and 525 nm for emission. As controlslactobacilli alone as well as stained mutans Streptococci were treatedand measured as described. The background fluorescence measured for therespective mutans Streptococci alone was subtracted from the valuemeasured for the aggregation with the respective lactobacillus. Allmeasurements were done in triplicate. The mutans Streptococci wereaggregated by all tested lactobacilli (see FIG. 3).

EXAMPLE 6 Specificity of the Aggregation Towards Typical Members of theOral Flora

The Lactobacillus cultures were grown as described in Example 1. Theoral bacteria namely: Streptococcus salivarius subsp. thermophilus(isolated by OrganoBalance, identified by API 50 CH (Biomerieux, France)according to manufacturer's instructions); Streptococcus oralis (DSMZ20066); Streptococcus oralis (DSMZ 20395); Streptococcus oralis (DSMZ20627); Staphylococcus epidermidis (DSMZ 1798); Staphylococcusepidermidis (DSMZ 20044); Streptococcus mitis (DSMZ 12643);Streptococcus sanguinis (DSMZ 20567)—were grown in 5 ml BHI-medium inclosed 15 ml Falcon tubes at 37° C. over night. Each of the abovementioned oral bacteria was preferably mixed in a volumetric ratio of3:1 with Lactobacillus cultures and aggregation was assayed as inExample 4. For each testing of aggregation/non-aggregation only one ofthe aforementioned bacteria is preferably used to immediately determinethe outcome of the testing.

As a control, a self-aggregation of the respective oral bacteria as wellas the tested Lactobacillus strains was always investigated byperforming the test with only the lactobacilli or the oral flora strainsadded to the tube.

The L. paracasei ssp. paracasei strains Lb-OB-KI (DSMZ 16667), Lb-OB-K2(DSMZ 16668), Lb-OB-K3 (DSMZ 16669), Lb-OB-K4 (DSMZ 16670), Lb-OB-K5(DSMZ 16671), did not aggregate the oral bacteria mentioned above. Theyare thus “specifically binding to mutans Streptococci” according to theabove definition. The L. rhamnosus strains LbOB-K6 (DSMZ 16672) andLb-OB-K7 (DSMZ 16673) aggregated Streptococcus salivarius subspthermophilus. They are nevertheless considered “specifically binding tomutans Streptococci” according to the above less preferred definition.

BHI-Broth: BHI-mixture (Difco, USA) 37 g/L pH: 7.2 EXAMPLE 7 TemperatureResistance of the Aggregating Capacity of the Lactobacilli

The bacteria were grown as in Example 1. The grown lactobacilli cultureswere autoclaved at 121° C. at 2 bar in satured steam for 20 min. Aftercooling of the autoclaved cultures to room temperature, the lactobacilliwere mixed in a volumetric ratio of 1:3 with grown S. mutans culturesand aggregation was assayed as in Example 4 including the controlexperiments. Aggregation was also assayed using the oral bacteria asoutlined in Example 6.

It was found that the aggregation behaviour of the lactobacilli was notchanged by the autoclaving procedure towards the tested S. mutansserotypes or towards the oral bacteria.

EXAMPLE 8 Aggregation by Heat-Inactivated Lactobacilli

The lactobacilli were grown as described in Example 1. MutansStreptococci were grown and stained as described in Examples 1 and 3.The grown lactobacilli cultures were adjusted to an OD600 of 2 asdescribed in Example 1. 1 ml of that suspension was autoclaved at 121°C. at 2 bar for 20 min as described above. After cooling of theautoclaved cultures to room temperature, aggregation was measured asdescribed in Example 5 including control experiments. Theheat-inactivated lactobacilli still aggregated all mutans Streptococci.

EXAMPLE 9 Dependency of the Aggregation on pH-Value

The bacteria were grown as in Example 1. 0.5 ml of the lactobacilli and1.5 ml of S. mutans were harvested by centrifugation at 3200×g for 10min and the supernatant was discarded. The cells were resuspended intheir original volume (0.5 ml and 1.5 ml, respectively) in differentPBS-buffers adjusted to different pH-values. The pH-values of thebuffers were adjusted to values from 7.0 to 3.0 in steps of 0.5pH-units. Cultures were resuspended in buffers of the respectivepH-value that was to be used for the aggregation behaviour assay.

Afterwards the lactobacilli were mixed in a volumetric ratio of 1:3 withS. mutans cultures and aggregation was assayed as in Example 4 includingthe control experiments. No visible aggregation of S. mutans by thelactobacilli occurred at pH values lower than 4.5.

EXAMPLE 10 Dependency of the Aggregation on pH-Value

The lactobacilli were grown as described in Example 1. MutansStreptococci were grown and stained as described in Examples 1 and 3.Afterwards the aggregation was assayed in different pH-values. For thispurpose lactobacilli as well as streptococci were resuspended in acetatebuffer adjusted to the respective pH. pH values tested were 4.0, 4.5 and5.0. The aggregation was assayed as described in Example 5. Noaggregation of mutans Streptococci occurred at pH values lower than 4.5.

EXAMPLE 11 Sensitivity of the Aggregation Behaviour to Lyophilisation

The bacteria were grown as in Example 1. Aliquots of 1 ml of thelactobacilli cultures were harvested by centrifugation at 3200×g for 10minutes. The supernatant was discarded and the pellets were lyophilisedat room temperature under vacuum for two hours. Resulting dry pellets ofeach tested Lactobacillus strain were stored at room temperature and at4° C., respectively, for 1 day, 1 week, 2 weeks, 3 weeks and 4 weeks.After the storage time, lyophilised pellets were resuspended in 1 mlPBS-buffer, pH 7.0. The resuspended lactobacilli were mixed in avolumetric ratio of 1:3 with freshly grown S. mutans cultures andaggregation was assayed as in Example 4 including the controlexperiments.

The aggregation behaviour of the mentioned lactobacilli towards S.mutans was not changed by the lyophilization or the storage procedures

EXAMPLE 12 Sensitivity of the Aggregation Behaviour to Lyophilisation

The lactobacilli were grown as described in Example 1. MutansStreptococci were grown and stained as described in Examples 1 and 3.The grown lactobacilli cultures were adjusted to an OD600 of 2 asdescribed in Example 1. 1 ml of that suspension was lyophilized at roomtemperature under vacuum for two hours. Afterwards, the lyophilisedpellets were resuspended in 1 ml PBS-buffer. Aggregation was measured asdescribed in Example 5, including control experiments.

The aggregation behaviour of the mentioned lactobacilli towards mutansStreptococci was not changed by the lyophilization.

EXAMPLE 13 Test on Protease Resistance

The bacteria were grown as in Example 1. Proteases used were Pronase E,Proteinase K, Trypsin, Chymotrypsin (all obtained from Sigma, Germany).Aliquots of 1 ml of the lactobacilli were washed in PBS-buffer byharvesting the cells by centrifugation at 3200×g for 10 minutes andresuspending the pellet in 1 ml PBS-buffer (pH 7.0). Afterwards thecells were harvested again as described above and resuspended inPBS-buffer (pH 7.0) containing the respective protease at a finalconcentration of 2.5 mg/ml. The suspension was incubated for 1 hour at37° C. Afterwards the cells were washed and resuspended in PBS-buffer(pH 7.0) as described above.

The aggregation was assayed as in Example 3 including the controlexperiments. The aggregation behaviour of the mentioned lactobacillitowards S. mutans was not changed by treatment with any of the mentionedproteases.

EXAMPLE 14 Protease Susceptibility of Aggregation Behaviour of theLactobacilli

The lactobacilli were grown as described in Example 1. MutansStreptococci were grown and stained as described in Examples 1 and 3.Used proteases were Pronase E, Proteinase K, Trypsin, Chymotrypsin (allobtained from Sigma, Germany). Aliquots of 1 ml of the lactobacilli werewashed in PBS-buffer by harvesting the cells by centrifugation at 3200×gfor 10 min and resuspending the pellet in 1 ml PBS-buffer (pH 7.0).Afterwards, the cells were harvested again as described above andresuspended in PBS-buffer (pH 7.0) containing the respective protease ata final concentration of 2.5 mgl/ml. The suspension was incubated for 1hour at 37° C. Afterwards, the cells were washed and resuspended inPBS-buffer (pH 7.0) as described above. The aggregation was assayed asdescribed in Example 5 including control experiments. The aggregationbehaviour of the lactobacilli towards mutans Streptococci was notchanged by the treatment with any of the mentioned proteases.

EXAMPLE 15 Ion Dependency of the Aggregation Behaviour

The bacteria were grown as in Example 1. Aliquots of 1 ml of thelactobacilli were washed in 1 ml 200 mM EDTA solution twice as describedabove. Afterwards the cells were harvested and resuspended in 1 mlPBS-buffer (pH 7.0).

The aggregation was assayed as in Example 4 and a complete loss of theaggregation ability was observed. Resuspension of the lactobacilli in 1ml of a 2 mM calcium solution after the two times washing in 200 mMEDTA-solution restored the ability to aggregate S. mutans. Resuspensionof the EDTA washed cells in up to 100 mM magnesium solution did notrestore the ability to aggregate S. mutans.

EXAMPLE 16 Ion Dependency of the Aggregation Behaviour

The lactobacilli were grown as described in Example 1. MutansStreptococci were grown and stained as described in Examples 1 and 3.Aliquots of 1 ml of the lactobacilli were washed in 1 ml 200 mM EDTAsolution twice as described above. Afterwards the cells were harvestedand resuspended in 1 ml PBS-buffer (pH 7.0). The aggregation was assayedas described in Example 5 and a complete loss of the aggregation abilitywas observed. Resuspension of the lactobacilli in 1 ml of a 2 mM calciumsolution after the two times washing in 200 mM EDTA-solution restoredthe ability to aggregate S. mutans. Resuspension of the EDTA washedcells in up to 100 mM magnesium solution did not restore the ability toaggregate mutans Streptococci.

EXAMPLE 17 Test of Aggregation in the Presence of Saliva

The bacteria were grown as in Example 1. 2 ml aliquots of S. mutanscultures were harvested as described above and resuspended in 2 ml ofsaliva. The saliva was provided by two volunteers and used immediatelyafter winning. The aggregation was assayed as in Example 4.

The aggregation behaviour of the mentioned lactobacilli towards S.mutans did not change in the presence of saliva.

EXAMPLE 18 Aggregation of Mutans Streptococci in the Presence of Saliva

Fresh saliva was sampled from volunteers. Saliva-flow was induced bychewing of sugar-free chewing gum. Volunteers collected 15 ml salivawith each sampling. The freshly collected saliva was diluted 1:2 withPBS-buffer for the assay procedure. Lactobacilli and mutans Streptococciwere cultivated as described in Example 1. Mutans Streptococci werestained as described in Example 3, except that after the stainingprocedure the stained cells were resuspended in saliva, instead ofPBS-buffer. The aggregation was measured as described in Example 5including control experiments. The presence of saliva did not inhibitthe aggregation.

Isolation Example 1 Obtaining of Further Lactobacilli

Lactobacilli can be obtained from any source, e.g. by oral cavity swabsof volunteers with low amount of caries and/or dental calculus. Also,Lactobacilli can be obtained from cell culture collections like DSMZ andATCC, or from other known sources of lactic acid bacteria, e.g. plants,foods and feeds. Strains obtained are then purified by standardmicrobiological techniques. Selective media for Lactobacilli are forexample described by Rogosa et al. 1951. A selective medium for theisolation of oral and fecal lactobacilli, J. Bacteriol. 62: 132-133.

Isolated strains are grown as described in Example 1. Selection forbinding is performed as described in Isolation Example 2.

Isolation Example 2

Stained Streptococcus mutans (DSMZ 20523) was prepared and distributedto 96 well microtiter plates as described above in Example 5. Also asdescribed in Example 5, 50 μl of the strains to be screened for bindinggrown according to example 1 were added to each microtiter plate welland vortexed at full speed for 12 minutes. Afterwards, the plate wascentrifuged at 500×g for 10 seconds and supernatant was carefullyremoved. The pellet was resuspended in 100 μl PBS and fluorescence ofthe suspension was measured as described in Example 5.

The strains to be screened were then tested as described above inExample 5 for binding to S. mutans DSMZ 20523, S. sobrinus DSMZ 20742,S. cricetus DSMZ 20562, S. ratti DSMZ 20564, S. ferus DSMZ 20646 and S.macacae DSMZ 20724. Positive binders were found to produce aggregates.

Food and Feed Example 1 Dog Puppy Feed

Main Ingredients: beef, brown rice, canola seed, flax seed meal,sunflower seed, buckwheat seed (soba), barley, millet.

Lesser Ingredients: carrots, red beets, broccoli, high-oleic sunfloweroil, canola oil, sea salt, oregano, garlic, a scorbic acid, propolis,vitamins (vitamins A, B12, D3, C, E, thiamine, riboflavin, pyridoxine,biotin, folic acid, pantothenate, niacin), minerals (chrome, sodiumselenite, iron, cooper citrate, zinc sulfate, cobalt).

To 1 kg of a base composition having the above main and lesseringredients, 3 g of dried autoclaved binder microorganisms and fragmentsthereof were added. The microorganism material was prepared by growingthe binder microorganism as described in Example 1. That is,lactobacilli were grown in MRS-medium. 5 ml MRS-medium were inoculatedwith 10 μl of the stock and incubated for 3 days at 37° C. under aerobicconditions. The microorganisms were pelleted by centrifugation andwashed with PBS once. The resuspended microorganisms were autoclaved asdescribed in Example 8. The autoclaved microorganisms were dried in anoven at 75° C. over night to obtain the dried heat-inactivated bindermicroorganisms and fragments thereof. The dog puppy food was preparedwith each of the deposited Lactobacillus strains DSMZ 16667, 16668,16669, 16670, 16671, 16672 and 16673 separately to obtain 7 separatepuppy foods.

The total content of the following parts is (in wt.-% of the totalfeed):

Protein (min) 26.0Fat (min) 14.0Fiber (max) 5.0Moisture (max) 10.5

Adult Dog Feed

Main Ingredient: beef, brown rice, canola seed, flax seed meal,sunflower seed, buckwheat seed (soba), barley, millet.

Lesser Ingredients: carrots, red beets, broccoli, high-oleic sunfloweroil, canola oil, sea salt, oregano, garlic, ascorbic acid, propolis,vitamins (i.e. vitamins A, B12, D3, C, E, thiamine, riboflavin,pyridoxine, biotin, folic acid, pantothenate, niacin), minerals (chrome,sodium selenite, iron, cooper citrate, zinc sulfate, cobalt).

As described for the dog puppy food, 7 adult dog food preparations wereprepared using the deposited Lactobacillus strains DSMZ 16667, 16668,16669, 16670, 16671, 16672 and 16673 separately.

The total content of the following parts is (in wt.-% of the totalfeed):

Protein (min) 20.0Fat (min) 12.0Fiber (max) 5.0Moisture (max) 10.5

Vegetarian Dog Feed.

Main Ingredients: soymeal, brown rice, canola seed, flax seed meal,sunflower seed, buckwheat seed, barley, millet,

Lesser Ingredients: carrots, red beets, broccoli, high-oleic sunfloweroil, canola oil, sea salt, oregano, garlic, ascorbic acid, propolis,vitamins (i.e. vitamins A, B12, D3, C, E, thiamine, riboflavin,pyridoxine, biotin, folic acid, pantothenate, niacin), minerals (i.e.chrome, sodium selenite, iron, cooper citrate, zinc sulfate, cobalt).

As described for the dog puppy food, 7 vegetarian dog food preparationswere prepared using the deposited Lactobacillus strains DSMZ 16667,16668, 16669, 16670, 16671, 16672 and 16673 separately.

The total content of the following parts is (in wt.-% of the totalfeed):

Protein (min) 20.0Fat (min) 12.0Fiber (max) 9.0Moisture (max) 10.5.

All food preparations were fed to a separate group of 5 dogs of theappropriate age (1-8 months, 2-4 years and 2-4 years, respectively) onceper day. As required by the dogs, other standard feed was provided atother times of the day. However, except for the above food preparationsaccording to the invention no nother feed was provided that wasadvertised by the respective manufacturer as particularly promoting oralhealth. After 6 months, none of the dogs had developed dental calculus.Also, oral malodor of the dogs measured by 10 volunteers 3 h after lastfeeding was considered tolerable.

Food and Feed Example 2 Dog Chew Product

A strip of cow rawhide was dunked in a culture of binder microorganismstrains DSMZ 16667, 16668, 16669, 16670, 16671, 16672 and 16673,respectively. The binder microorganisms were grown as described in Foodand Feed Example 1. The rawhide strips were then formed into a bone-likeshape and autoclaved as described in Food and Feed Example 1.

The autoclaved strips were air dried. The dried bone-like strips weregiven to 5 dogs of 5-8 years which just had their dental calculusremoved. New dried bone-like strips were then given twice per week.Except for the dried bone-like strips, the dogs were fed as beforeremoval of dental calculus. Within 8 months, none of the dogs developeddental calculus again. Also, oral malodor of the dogs measured by 10volunteers 3 h after last feeding was considered tolerable.

Food and Feed Example 3 Further Dog Feeds Puppy Feed:

Main ingredients: chicken, chicken meal, barley, peas, brown rice,ground extruded whole soybeans, chicken fat (preserved with mixedtocopherols).

Lesser ingredients: salmon meal, natural chicken liver flavor, brewersdried yeast, flaxseed meal, dried eggs, apples, carrots, potassiumchloride, dicalcium phosphate, minerals (i.e. zinc proteinate, ironproteinate, copper proteinate, manganese proteinate, sodium selenite,cobalt proteinate, calcium iodate), salt, choline chloride, vitamins(i.e. vitamin E supplement, 1-ascorbyl-2-polyphosphate, vitamin B12supplement, d-calcium pantothenate, vitamin A supplement, niacin,riboflavin, folic acid, biotin, pyridoxine hydrochloride, thiaminemononitrate, vitamin D3 supplement, calcium carbonate), yeast culture(saccharomyces cerevisiae), dried enterococcus faecium fermentationproduct, dried lactobacillus acidophilus fermentation product, driedaspergillus niger fermentation extract, trichoderma longibrachiatumfermentation extract, dried bacillus subtilis fermentation extract andfermentation solubles

The total content of the following parts is (in wt.-% of the totalfeed):

Protein wt.-% (min): 28.0Fat wt.-% (min): 15.0Fiber wt.-% (max): 3.5Moisture wt.-% (max): 10.0.

Adult Dog Feed:

Main ingredients: chicken, chicken meal, barley, peas, oats, brown rice,chicken fat (preserved with mixed tocopherols).

Lesser ingredients: natural chicken liver flavor, brewers dried yeast,salmon meal, dried eggs, apple, whole flaxseed, carrots, dicalciumphosphate, potassium chloride, salt, minerals (i.e. zinc proteinate,iron proteinate, copper proteinate, manganese proteinate, sodiumselenite, cobalt proteinate, calcium iodate), choline chloride, vitamins(i.e. vitamin E supplement, 1-ascorbyl-2-polyphosphate, vitamin B12supplement, d-calcium pantothenate, vitamin A supplement, niacin,riboflavin, folic acid, biotin, pyridoxine hydrochloride, thiaminemononitrate, vitamin D3 supplement), glucosamine hydrochloride,chondroitin sulfate, yeast culture (saccharomyces cerevisiae), driedenterococcus faecium fermentation product, dried lactobacillusacidophilus fermentation product, dried aspergillus niger fermentationextract, dried trichoderma longibrachiatum fermentation extract, driedbacillus subtilis fermentation extract and fermentation solubles.

The total content of the following parts is (in wt.-% of the totalfeed):

Protein wt.-% (min): 26.0Fat wt.-% (min): 13.0Fiber wt.-% (max): 3.5Moisture wt.-% (max): 10.0.

Weight Control Dog Feed:

Main ingredients: brown rice, chicken meal, barley, oats, peas, chicken,chicken fat (preserved with mixed tochopherols).

Lesser ingredients: natural chicken liver flavor, brewers dried yeast,salmon meal, dried eggs, apple, whole flaxseed, carrots, dicalciumphosphate, potassium chloride, salt, minerals (i.e. zinc proteinate,iron proteinate, copper proteinate, manganese proteinate, sodiumselenite, cobalt proteinate, calcium iodate), choline chloride, vitamins(i.e. vitamin E supplement, 1-ascorbyl-2-polyphosphate, vitamin B12supplement, d-calcium pantothenate, vitamin A supplement, niacin,riboflavin, folic acid, biotin, pyridoxine hydrochloride, thiaminemononitrate, vitamin D3 supplement), glucosamine hydrochloride,chondroitin sulfate, yeast culture (saccharomyces cerevisiae), driedenterococcus faecium fermentation product, dried lactobacillusacidophilus fermentation product, dried aspergillus niger fermentationextract, dried trichoderma longibrachiatum fermentation extract, driedbacillus subtilis fermentation extract and fermentation solubles.

The total content of the following parts is (in wt.-% of the totalfeed):

Protein wt.-% (min): 24.0Fat wt.-% (min): 10.0Fiber wt.-% (max): 3.5Moisture wt.-% (max): 10.0.

As described for the dog puppy food of Food and Feed Example 1, 7corresponding puppy, adult and weight control dog food preparations wereprepared using the deposited Lactobacillus strains DSMZ 16667, 16668,16669, 16670, 16671, 16672 and 16673 separately.

High Protein Adult Dog Feed:

Main Ingredients: De-boned chicken, chicken meal, turkey meal, russetpotato, lake whitefish, chicken fat (preserved with mixed tocopherols).

Lesser Ingredients: Sweet potato, whole eggs, turkey, salmon meal,salmon and anchovy oils, salmon, natural chicken flavor, sunflower oil,sun-cured alfalfa, dried brown kelp, carrots, spinach, peas, tomatoes,apples, psyllium, dulse, chicory root, licorice root, tumeric root,fenugreek, glucosamine HCl, cranberries, black currants, marigoldflowers, L-carnitine, sweet fennel, zea mays, peppermint leaf, chamomileflowers, dandelion, summer savory, rosemary extract, chondroitinsulfate, rosehips, vitamins (i.e. vitamin E, choline chloride, vitaminA, vitamin D3, thiamine mononitrate, vitamin B12, folic acid, biotin),sea salt, minerals (i.e. iron proteinate, zinc proteinate, manganeseproteinate, copper proteinate), dried Lactobacillus acidophilusfermentation product, dried Enterococcus faecium fermentation product.

The total content of the following parts is (in wt.-% of the totalfeed):

Protein min: 42 wt.-% Fat min: 16 wt.-% Fiber max 3 wt.-% Moisture max:10 wt.-% Ash max: 7 wt.-%.

As described for the dog puppy food of Food and Feed Example 1, 7corresponding dog feed preparations were prepared using the depositedLactobacillus strains DSMZ 16667, 16668, 16669, 16670, 16671, 16672 and16673 separately.

Vegetarian Dog Feed:

Main Ingredients: brown rice, canola seed, flax seed meal, sunflowerseed, buckwheat seed (soba), barley, millet, carrots, red beets,broccoli, high-oleic sunflower oil.

Lesser Ingredients: canola oil, sea salt, oregano, garlic, ascorbicacid, propolis, vitamins (i.e. vitamins A, B12, D3, C, E, K3, thiamine,riboflavin, pyridoxine, biotin, folic acid, pantothenate, niacin),minerals (i.e. chrome, sodium selenite, iron, cooper citrate, zincsulfate, cobalt).

The total content of the following parts is (in wt.-% of the totalfeed):

Protein min: 20 wt.-% Fat min: 12 wt.-% Fiber max: 9 wt.-% Moisture max:10.5 wt.-%.

As described for the dog puppy food of Food and Feed Example 1, 7corresponding dog feed preparations were prepared using the depositedLactobacillus strains DSMZ 16667, 16668, 16669, 16670, 16671, 16672 and16673 separately.

FURTHER EMBODIMENTS

-   1. Binder microorganism or fragment thereof, wherein the    microorganism or fragment thereof is capable of binding to at least    one, preferably at least two and more preferably at least three    Streptococcus strain of the mutans Streptococcus group, and wherein    the binding is resistant to heat treatment and/or resistant to    protease treatment and/or is calcium dependent and/or is formed    within a pH above 4.0 and/or is independent of magnesium and/or    formed in the presence of saliva as an oral care agent.-   2. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is resistant to heat treatment at 55° C.,    preferably at 65° C., more preferably at 95-121° C. and most    preferably at 121° C.-   3. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is resistant to heat treatment for 15 min,    preferably 15-120 min, preferably 15-30 min and most preferably 20    min.-   4. Binder microorganism or fragment thereof according to embodiment    3, wherein the binding is resistant to heat treatment in saturated    steam at a pressure of 1-5 bar, preferably 1-3 bar, more preferably    2 bar.-   5. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is resistant to heat treatment in saturated    steam at a pressure of 1-5 bar, preferably 1-3 bar, more preferably    2 bar, at a temperature of 95-121° C., more preferably at 121° C.,    for 15 min, preferably 15-120 min, preferably 15-30 min and most    preferably 20 min.-   6. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is resistant to heat treatment, wherein the    heat treatment is in saturated steam at a pressure of 2 bar at a    temperature of 121° C. for 20 min.-   7. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is resistant to protease treatment by pronase    E, proteinase K, trypsin and/or chymotrypsin, and preferably is also    resistant to protease treatment by elastase, thrombin,    aminopeptidase I, carboxypeptidase, dostripain, endoproteinase,    papain, cathepsin B, pepsin, gastricsin, chymosin and/or cathepsin    D.-   8. Binder microorganism or fragment thereof according to embodiment    7, wherein the binding is resistant to protease treatment by pronase    E, proteinase K, trypsin and chymotrypsin.-   9. Binder microorganism or fragment thereof according to embodiment    7, wherein the binding is resistant to protease treatment by    elastase, thrombin, aminopeptidase I, carboxypeptidase, dostripain,    endoproteinase, papain, cathepsin B, pepsin, gastricsin, chymosin    and cathepsin D.-   10. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is resistant to heat treatment in saturated    steam at a pressure of 1-5 bar, preferably 1-3 bar, more preferably    2 bar, at a temperature of 95-121° C., more preferably at 121° C.,    for 15 min, preferably 15-120 min, preferably 15-30 min and most    preferably 20 min; and also is resistant to protease treatment by    pronase E, proteinase K, trypsin and/or chymotrypsin, and preferably    is also resistant to protease treatment by elastase, thrombin,    aminopeptidase I, carboxypeptidase, dostripain, endoproteinase,    papain, cathepsin B, pepsin, gastricsin, chymosin and/or cathepsin    D.-   11. Binder microorganism or fragment thereof according to embodiment    6, wherein the binding is resistant to protease treatment by pronase    E, proteinase K, trypsin and/or chymotrypsin, and preferably is also    resistant to protease treatment by elastase, thrombin,    aminopeptidase I, carboxypeptidase, dostripain, endoproteinase,    papain, cathepsin B, pepsin, gastricsin, chymosin and/or cathepsin    D.-   12. Binder microorganism or fragment thereof according to embodiment    8, wherein the binding is resistant to heat treatment, wherein the    heat treatment is in saturated steam at a pressure of 2 bar at a    temperature of 121° C. for 20 min.-   13. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is dependent on the presence of Ca2+ ions in    a concentration of at least 2 mM, more preferably at least 1 mM,    most preferably at least 0.05 mM.-   14. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is dependent on the presence of Ca2+ ions in    a concentration of 0.05-500 mM, more preferably 1-100 mM, most    preferably 2-30 mM.-   15. Binder microorganism or fragment thereof according to embodiment    12 or 13, wherein the binding is resistant to heat treatment,    wherein the heat treatment is in saturated steam at a pressure of 2    bar at a temperature of 121° C. for 20 min.-   16. Binder microorganism or fragment thereof according to embodiment    12 or 13, wherein the binding is resistant to protease treatment by    pronase E, proteinase K, trypsin and/or chymotrypsin, and preferably    is also resistant to protease treatment by elastase, thrombin,    aminopeptidase I, carboxypeptidase, dostripain, endoproteinase,    papain, cathepsin B, pepsin, gastricsin, chymosin and/or cathepsin    D.-   17. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is formed at a pH of 4.0-9.0, preferably    4.0-7.0, more preferably 4.2-5.0 and most preferably 4.5.-   18. Binder microorganism or fragment thereof according to embodiment    16, wherein the binding is resistant to heat treatment, wherein the    heat treatment is in saturated steam at a pressure of 2 bar at a    temperature of 121° C. for 20 min.-   19. Binder microorganism or fragment thereof according to embodiment    16, wherein the binding is resistant to protease treatment by    pronase E, proteinase K, trypsin and/or chymotrypsin, and preferably    is also resistant to protease treatment by elastase, thrombin,    aminopeptidase I, carboxypeptidase, dostripain, endoproteinase,    papain, cathepsin B, pepsin, gastricsin, chymosin and/or cathepsin    D.-   20. Binder microorganism or fragment thereof according to embodiment    16, wherein the binding is dependent on the presence of Ca2+ ions in    a concentration of at least 2 mM, more preferably at least 1 mM,    most preferably at least 0.05 mM.-   21. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is independent of the presence of magnesium    ions, and preferably is resistant to heat treatment, wherein the    heat treatment is in saturated steam at a pressure of 2 bar at a    temperature of 121° C. for 20 min.-   22. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is formed in the presence of saliva, and    preferably is resistant to heat treatment, wherein the heat    treatment is in saturated steam at a pressure of 2 bar at a    temperature of 121° C. for 20 min.-   23. Binder microorganism or fragment thereof according to embodiment    1, wherein the binding is resistant to heat treatment, resistant to    protease treatment, and is formed in the presence of saliva.-   24. Binder microorganism or fragment thereof according to any of the    previous embodiments, wherein the strain of mutans Streptococcus is    selected from the group consisting of Streptococcus mutans serotype    c (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923),    Streptococcus mutans serotype f (NCTC 11060), Streptococcus sobrinus    DSMZ 20742, Streptococcus ratti DSMZ 20564, Streptococcus cricetus    DSMZ 20562, Streptococcus ferus DSMZ 20646 and Streptococcus macacae    DSMZ 20714, and preferably wherein the specific binding of the    binder microorganism or fragment thereof can be assayed as follows:    -   (a) growing said binder microorganism to stationary phase, or,        in case a fragment is to be tested, obtaining such fragment,    -   (b) mixing said binder microorganism or fragment with a mutans        Streptococcus which has been grown to stationary phase,    -   (c) incubating the mixture obtained in step (b) under conditions        allowing the formation of aggregates of said microorganism and        said Streptococcus, and    -   (d) detecting aggregates by the occurrence of a pellet.-   25. Binder microorganism or fragment thereof according to any of    embodiments 1-22,

wherein the microorganism or fragment thereof is capable of binding toeach of the strains selected from the group consisting of Streptococcusmutans serotype c (DSMZ 20523), Streptococcus mutans serotype e (NCTC10923) and Streptococcus mutans serotype f (NCTC 11060).

-   26. Binder microorganism or fragment thereof according to embodiment    1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,    20, 21, 22, 23 or 24, wherein the microorganism or fragment thereof    is not capable of binding to at least one, preferably at least two    and more preferably at least three and even more preferably all    microorganisms selected from the group consisting of Streptococcus    salivarius ssp. thermophilus, Streptococcus oralis DSMZ 20066,    Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627,    Streptococcus mitis DSMZ 12643 and Streptococcus sanguinis DSMZ    20567.-   27. Binder microorganism or fragment thereof according to embodiment    1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,    20, 21, 22, 23, 24 or 25, wherein the binder microorganism is of    family Lactobacillaceae, preferably of genus Lactobacillus, genus    Paralactobacillus, genus Pediococcus or genus Sharpea, more    preferably of species Lactobacillus paracasei, species Lactobacillus    rhamnosus, species Lactobacillus casei or species Lactobacillus    zeae, and most preferably is any of strains DSMZ 16667, DSMZ 16668,    DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 or DSMZ 16673, or a    mutant or derivative thereof.-   28. Binder microorganism or fragment thereof according to embodiment    26, wherein the microorganism or fragment thereof is capable of    binding to each of the strains selected from the group consisting of    Streptococcus mutans serotype c (DSMZ 20523), Streptococcus mutans    serotype e (NCTC 10923) and Streptococcus mutans serotype f    (NCTC 11060) and is not capable of binding to a microorganism    selected from the group consisting of Streptococcus salivarius ssp.    thermophilus, Streptococcus oralis DSMZ 20066, Streptococcus oralis    DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis    DSMZ 12643 and Streptococcus sanguinis DSMZ 20567.-   29. Binder microorganism or fragment thereof according to embodiment    26 or 27, wherein the microorganism or fragment thereof, after heat    treatment in saturated steam at a pressure of 2 bar at a temperature    of 121° C. for 20 min, retains the capability to bind to a mutans    Streptococcus selected from the group consisting of Streptococcus    mutans serotype c (DSMZ 20523), Streptococcus mutans serotype e    (NCTC 10923), Streptococcus mutans serotype f (NCTC 11060),    Streptococcus sobrinus DSMZ 20742, Streptococcus ratti DSMZ 20564,    Streptococcus cricetus DSMZ 20562, Streptococcus ferus DSMZ 20646    and Streptococcus macacae DSMZ 20714 in the presence of at least    0.05 mM calcium ions and in the presence of saliva and independent    of a treatment by pronase E, proteinase K, trypsin and/or    chymotrypsin, wherein under these conditions the heat treated    microorganism or fragment thereof is not capable of binding to a    microorganism selected from the group consisting of Streptococcus    salivarius ssp. thermophilus, Streptococcus oralis DSMZ 20066,    Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627,    Streptococcus mitis DSMZ 12643 and Streptococcus sanguinis DSMZ    20567.-   30. Binder microorganism according to any of embodiments 1, 2, 3, 4,    5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,    23, 24, 25, 26, 27, 28, 29 or 30, wherein the binder microorganism    is in an inactivated form obtainable or obtained by thermal    inactivation, lyphilisation or spray drying, wherein thermal    inactivation is preferably achieved by    -   autoclaving cells of said binder microorganism at a temperature        of 121° C. for at least 20 min in the presence of saturated        steam at an atmospheric pressure of 2 bar, or    -   freezing said cells for at least 1 h at −20° C.-   31. Inactivated binder microorganism according to embodiment 29,    obtainable or obtained by thermal inactivation at a temperature of    ≧55° C., preferably at a temperature of ≧65° C., even more    preferably at a temperature of 95-121° C. and most preferably at a    temperature of 121° C.-   32. Inactivated binder microorganism according to embodiment 30,    obtainable or obtained by thermal inactivation for ≧15 min,    preferably 15-120 min, preferably 15-30 min and most preferably 20    min.-   33. Inactivated binder microorganism according to embodiment 29, 30    or 31, obtainable or obtained by thermal inactivation in saturated    steam at a pressure of 1-5 bar, preferably 1-3 bar, more preferably    2 bar, and most preferably by thermal inactivation in saturated    steam at a pressure of 2 bar at a temperature of 121° C. for 20 min.-   34. Fragment of a binder microorganism according to any of the    previous embodiments, wherein the fragment is a lysate or membrane    fragment of a binder microorganism.-   35. Binder microorganism or fragment thereof according to any of    embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,    17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or    33, as a sensorically neutral oral care agent.-   36. Binder microorganism or fragment thereof according to any of    embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,    17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or    33, as an anti-dental calculus agent, sensorically neutral    anti-caries agent and/or anti-oral malodor agent.-   37. Binder microorganism or fragment thereof according to embodiment    35, as a sensorically neutral anti-dental calculus agent,    sensorically neutral anti-caries agent and/or sensorically neutral    anti-oral malodor agent.-   38. Binder microorganism or fragment thereof according to any of    embodiments 34, 35 or 36, wherein the binder microorganism or    fragment thereof is an isolated or purified binder microorganism or    fragment thereof.-   39. Composition comprising a binder microorganism or fragment    thereof according to any of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9,    10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,    27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 as an oral care agent,    preferably as an anti-dental calculus agent, an anti-caries agent    and/or an anti-oral malodor agent.-   40. Composition comprising a binder microorganism or fragment    thereof according to any of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9,    10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,    27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 as a sensorically    neutral oral care agent, preferably as a sensorically neutral    anti-dental calculus agent, a sensorically neutral anti-caries agent    and/or a sensorically neutral anti-oral malodor agent.-   41. Composition comprising a binder microorganism or fragment    thereof according to any of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9,    10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,    27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 in an amount    -   sufficient for preventing or reducing intensity of oral malodor,        and/or    -   sufficient for preventing caries or slowing down caries        generation and/or    -   sufficient for preventing dental calculus formation or slowing        down dental calculus formation.-   42. Composition comprising a binder microorganism according to any    of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,    16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,    34, 35, 36 or 37, in a concentration of    -   10²-10¹³ cells/mg, preferably 10²-10¹² cells/mg, even more        preferably 10³-10⁸ cells/mg, or        -   10²-10¹³ cells/ml, or    -   ≧0.01 wt.-% relative to the total composition, preferably        0.01-10 wt.-% and more preferably 0.025-2 wt.-%, or    -   ≧0.01 mg dry weight/g total composition, preferably 0.01-100 mg        dry weight/g total composition and more preferably 0.025-2 mg        dry weight/g total composition.-   43. Composition comprising a fragment of a binder microorganism    according to any of embodiments 32, 33, 34, 35, 36 or 37, in a    concentration of    -   ≧0.01 wt.-% relative to the total composition, preferably        0.01-10 wt.-% and more preferably 0.025-2 wt.-%, or    -   ≧0.01 mg dry weight/g total composition, preferably 0.01-100 mg        dry weight/g total composition and more preferably 0.025-2 mg        dry weight/g total composition.-   44. Composition according to any of embodiments 38, 39, 40, 41 or    42, comprising a further microorganism in a viable, a thermally    inactivated or lyphilized form, wherein the thermally inactivated    form is obtainable or obtained by treatment in saturated steam at a    pressure of 1-5 bar, preferably 1-3 bar, more preferably 2 bar, and    most preferably by thermal inactivation in saturated steam at a    pressure of 2 bar at a temperature of 121° C. for 20 min.-   45. Composition according to embodiment 43, wherein the further    microorganism is an anti-oral malodor microorganism in a    concentration sufficient for preventing, modifying or reducing oral    malodor, preferably capable of stimulating the growth of    Streptococcus salivarius but does not stimulate the growth of    Streptococcus mutans and/or Porphyromonas gingivalis.-   46. Composition according to embodiment 44, wherein the anti-oral    malodor microorganism is of species Lactobacillus acidophilus, and    preferably is any of the Lactobacillus strains DSMZ 19825, DSMZ    19826 or DSMZ 19827.-   47. Composition according to any of embodiments 38, 39, 40, 41, 42,    43, 44 or 45,    -   wherein the composition is for use with a human or animal, and        is a toothpaste, dentifrice, tooth powder, topical oral gel,        mouth rinse, denture product, mouthspray, lozenge, oral tablet,        chewing gum, mouth wash, dental floss, chew product or an        additive for food, feed or drinks, or    -   wherein the composition is in the form of a powder, tablet, film        preparation, solution, aerosol, granule, pill, suspension,        emulsion, capsule, syrup, liquid, elixir, extract, tincture or        fluid extract, sheet-like food, bottled food, canned food,        retort food or fluid food or    -   wherein the composition is a food or drink selected from the        group consisting of gum, spray, beverage, candy, infant formula,        ice cream, frozen dessert, sweet salad dressing, milk        preparation, cheese, quark, yogurt, acidified milk, coffee        cream, whipped cream, butter, cheese, processed milk and skimmed        milk, meat product—preferably ham, sausage, and hamburger, fish        meat, cake product, egg product—preferably seasoned egg rolls        and egg curd, confectionery—preferably cookie, jelly, snacks,        and chewing gum—, bread, noodles, pickle, smoked product, dried        fish, seasoning.-   48. Composition according to any of embodiments 38, 39, 40, 41, 42,    43, 44 or 45, wherein the composition is a food or feed composition.-   49. Food or feed composition according to embodiment 47, wherein the    composition is for an infant or for a pet animal, preferably a dog,    cat, rat, mouse, hamster, guinea pig or monkey.-   50. Pet food or feed composition according to embodiment 48, wherein    the composition is a pet chew product, and preferably is in the    shape of a bone, a roll, a donut, a bow, a pretzel, a figure eight,    or a chip.-   51. Composition according to any of embodiments 38, 39, 40, 41, 42,    43, 44, 45, 46, 47 or 48, wherein the composition is a cosmetic,    pharmaceutical or veterinary composition.-   52. Pet food or feed composition,    -   comprising an inactivated binder microorganism in a thermally        inactivated or lyphilized form, wherein the inactivated        microorganism is capable of binding to a Streptococcus strain of        the mutans Streptococcus group, and wherein the binding is        formed in the presence of saliva and is resistant to heat        treatment and calcium dependent, wherein the heat treatment is        in saturated steam at a pressure of 2 bar at a temperature of        121° C. for 20 min,    -   and wherein the mutans Streptococcus strain is selected from the        group consisting of Streptococcus mutans serotype c (DSMZ        20523), Streptococcus mutans sero-type e (NCTC 10923) and        Streptococcus mutans serotype f (NCTC 11060),    -   and wherein the inactivated binder microorganism thereof is not        capable of binding to a microorganism selected from the group        consisting of Streptococcus salivarius ssp. thermophilus,        Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ        20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ        12643 and Streptococcus sanguinis DSMZ 20567,    -   and wherein the binder microorganism is present in a        sensorically neutral amount        -   sufficient for preventing or reducing intensity of oral            malodor, and/or        -   sufficient for preventing caries or slowing down caries            generation and/or        -   sufficient for preventing dental calculus formation or            slowing down dental calculus formation.-   53. Pet food composition according to embodiment 51, wherein    concentration of inactivated binder microorganism is    -   10²-10¹³ cells/mg, preferably 10²-10¹² cells/mg, even more        preferably 10³-10⁸ cells/mg, or    -   10²-10¹³ cells/ml, or    -   ≧0.01 wt.-% relative to the total composition, preferably        0.01-10 wt.-% and more preferably 0.025-2 wt.-%, or    -   ≧0.01 mg dry weight/g total composition, preferably 0.01-100 mg        dry weight/g total composition and more preferably 0.025-2 mg        dry weight/g total composition.-   54. Pet food or feed composition, comprising a fragment according to    embodiment 33 in a concentration of    -   ≧0.01 wt.-% relative to the total composition, preferably        0.01-10 wt.-% and more preferably 0.025-2 wt.-%, or    -   ≧0.01 mg dry weight/g total composition, preferably 0.01-100 mg        dry weight/g total composition and more preferably 0.025-2 mg        dry weight/g total-   55. Use of a binder microorganism or fragment thereof according to    any of embodiments 1-37 as a sensorically neutral oral care agent,    preferably as a sensorically neutral anti-dental calculus agent    and/or sensorically neutral anti-caries agent and/or sensorically    neutral anti-oral malodor agent.-   56. Use of a binder microorganism or fragment thereof according to    any of embodiments 1-37 in the manufacture of any of    -   a medicament for prevention or treatment of dental calculus        formation,    -   a medicament for prevention or treatment of caries,    -   a medicament for prevention or treatment of oral malodor,        wherein the binder microorganism and/or fragment thereof is in a        sensorically neutral amount.

1. A method for providing oral care to a human or animal, comprisingadministering a composition comprising a binder microorganism orfragment thereof as a sensorically neutral oral care agent to a human oranimal in need thereof, wherein the binder microorganism is a lacticacid bacterium, wherein the binder microorganism or fragment thereof iscapable of binding to a microorganism of the group of mutansStreptococci, and wherein the binding is: (i) resistant to heattreatment; and/or (ii) resistant to protease treatment; and/or (iii)calcium-dependent; and/or (iv) formed within a pH range between 4.5 and8.5; and/or (v) formed in the presence of saliva; and/or (vi)independent of magnesium.
 2. A method for preparing a cosmetic,pharmaceutical or veterinary composition comprising formulating a bindermicroorganism or fragment thereof into a composition, wherein thecomposition is an oral care composition, wherein the bindermicroorganism is used in a sensorically neutral amount, wherein thebinder microorganism is a lactic acid bacterium, wherein the bindermicroorganism or fragment thereof is capable of binding to amicroorganism of the group of mutans Streptococci, and wherein thebinding is: (i) resistant to heat treatment; and/or (ii) resistant toprotease treatment; and/or (iii) calcium-dependent; and/or (iv) formedwithin a pH range between 4.5 and 8.5; and/or (v) formed in the presenceof saliva; and/or (vi) independent of magnesium.
 3. The method accordingto claim 2, wherein the binder microorganism or fragment thereof iscapable of binding to at least one strain, at least two strains, or atleast three strains of mutans Streptococci selected from the groupconsisting of Streptococcus mutans serotype c (DSMZ 20523),Streptococcus mutans serotype e (NCTC 10923), Streptococcus mutansserotype f (NCTC 11060), Streptococcus sobrinus DSMZ 20742,Streptococcus ratti DSMZ 20564, Streptococcus cricetus DSMZ 20562,Streptococcus ferus DSMZ 20646 and Streptococcus macacae DSMZ
 20714. 4.The method according to claim 2, wherein the binder microorganism orfragment thereof is not capable of binding to at least one, at leasttwo, at least three, or any microorganism selected from the groupconsisting of Streptococcus salivarius ssp. thermophilus, Streptococcusoralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralisDSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus sanguinisDSMZ
 20567. 5. The method according to claim 2, wherein the bindermicroorganism is in a thermally inactivated or lyophilized form, and/orwherein the binder microorganism is of the family Lactobacillaceae, ofthe genus Lactobacillus, Paralactobacillus, Pediococcus or Sharpea, ofspecies Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacilluscasei or Lactobacillus zeae, or is any of strains DSMZ 16667, DSMZ16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 or DSMZ 16673, ora mutant or derivative thereof.
 6. The method according to claim 2,wherein the binder microorganism or fragment thereof is used: as apreferably sensorically neutral anti-dental calculus agent, or for thepreparation of a composition for prevention or treatment of dentalcalculus, and/or as a sensorically neutral anti-caries agent, or for thepreparation of a sensorically neutral composition for prevention ortreatment of caries, and/or as a preferably sensorically neutralanti-oral malodor agent, or for the preparation of a composition forprevention or treatment of oral malodor.
 7. The method according toclaim 2, wherein the binder microorganism is selected from the groupconsisting of: microorganisms which metabolize D-lactose, but notL-sorbose and/or D-saccharose and/or D-inuline, (ii) microorganismswhich metabolize inuline, (iii) microorganisms which metabolizeL-sorbose, but not D-lactose and/or D-saccharose and/or inuline, and(iv) microorganisms which metabolize L-sorbose, D-lactose and inuline,and preferably, wherein the microorganism is selected from the groupconsisting of: (i) microorganisms which metabolize D-lactose, but notL-sorbose, D-saccharose and inuline, (ii) microorganisms whichmetabolize L-sorbose, D-lactose and inuline, but not D-saccharose, (iii)microorganisms which metabolize L-sorbose, but not D-lactose,D-saccharose and inuline, and (iv) microorganisms which metabolizesL-sorbose, D-lactose, D-saccharose, but not inuline.
 8. The methodaccording to claim 2, wherein the binding of said binder microorganismor fragment thereof to Streptococcus strains can be assayed as follows:(a) growing said microorganism to stationary phase; (b) mixing saidmicroorganism with said Streptococcus which has been grown to stationaryphase; (c) incubating the mixture obtained in step (b) under conditionsallowing the formation of aggregates of said microorganism and saidStreptococcus; and (d) detecting aggregates by the occurrence of apellet.
 9. A method of preparing a composition for prevention ortreatment of dental calculus and/or for the preparation of asensorically neutral composition for prevention or treatment of cariesand/or for the preparation of a composition for prevention or treatmentof oral malodor, comprising adding a binder microorganism or fragmentthereof to a base composition, wherein the binder microorganism is addedin a sensorically neutral amount, wherein the binder microorganism is alactic acid bacterium, wherein the binder microorganism is capable ofbinding to a microorganism of the group of mutans Streptococci, whereinthe binding is: (i) resistant to heat treatment; and/or (ii) resistantto protease treatment; and/or (iii) calcium-dependent; and/or (iv)formed within a pH range between 4.5 and 8.5; and/or (v) formed in thepresence of saliva; and/or (vi) independent of magnesium, and whereinthe base composition comprises a cosmetically, pharmaceutically orveterinary compatible carrier.
 10. The method according to claim 9,wherein the binder microorganism is in a thermally inactivated orlyophilized form, and/or wherein the binder microorganism or fragmentthereof is capable of binding to at least one strain, at least twostrains, or at least three strains of mutans Streptococci selected fromthe group consisting of Streptococcus mutans serotype c (DSMZ 20523),Streptococcus mutans serotype e (NCTC 10923) and Streptococcus mutansserotype f (NCTC 11060), Streptococcus sobrinus DSMZ 20742,Streptococcus ratti DSMZ 20564, Streptococcus cricetus DSMZ 20562,Streptococcus ferus DSMZ 20646 and Streptococcus macacae DSMZ 20714, andpreferably is not capable of binding to at least one, at least two, atleast three, or any microorganism selected from the group consisting ofStreptococcus salivarius ssp. thermophilus, Streptococcus oralis DSMZ20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627,Streptococcus mitis DSMZ 12643 and Streptococcus sanguinis DSMZ 20567,and/or wherein the binder microorganism is of family Lactobacillaceae,of the genus Lactobacillus, Paralactobacillus, Pediococcus or Sharpea,or of species Lactobacillus paracasei, Lactobacillus rhamnosus,Lactobacillus casei or Lactobacillus zeae, or is any of strains DSMZ16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 orDSMZ 16673, or a mutant or derivative thereof.
 11. A compositioncomprising an isolated or purified binder microorganism or isolated orpurified fragment thereof in an amount: sufficient for preventing dentalcalculus formation or slowing down dental calculus formation, and/orsufficient for preventing or reducing intensity of oral malodor, and/orsufficient for preventing caries or slowing down caries generation, andpreferably in a sensorically neutral amount, wherein the bindermicroorganism is a lactic acid bacterium, preferably wherein the bindermicroorganism is of family Lactobacillaceae, of genus Lactobacillus,Paralactobacillus, Pediococcus or Sharpea, of species Lactobacillusparacasei, Lactobacillus rhamnosus, Lactobacillus casei or Lactobacilluszeae, or is any of strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ16670, DSMZ 16671, DSMZ 16672 or DSMZ 16673, or a mutant or derivativethereof, and wherein the binder microorganism is capable of binding to amicroorganism of the group of mutans Streptococci, wherein the bindingis: (i) resistant to heat treatment; and/or (ii) resistant to proteasetreatment; and/or (iii) calcium-dependent; and/or (iv) formed within apH range between 4.5 and 8.5; and/or (v) formed in the presence ofsaliva; and/or (vi) independent of magnesium, and wherein preferably thetotal content of mono- and diglycerides is at most 20 wt.-%, at most 5wt.-%, or at most 2 wt.-% of the whole composition.
 12. The compositionaccording to claim 11, wherein the composition is for use with a humanor animal, and wherein the composition is a toothpaste, dentifrice,tooth powder, topical oral gel, mouth rinse, denture product,mouthspray, lozenge, oral tablet, chewing gum, mouth wash, dental floss,chew product or an additive for food, feed or drinks, or wherein thecomposition is in the form of a powder, tablet, film preparation,solution, aerosol, granule, pill, suspension, emulsion, capsule, syrup,liquid, elixir, extract, tincture or fluid extract, sheet-like food,bottled food, canned food, retort food or fluid food, or wherein thecomposition is a food or drink selected from the group consisting ofgum, spray, beverage, candy, infant formula, ice cream, frozen dessert,sweet salad dressing, milk preparation, cheese, quark, yogurt, acidifiedmilk, coffee cream, whipped cream, butter, cheese, processed milk andskimmed milk, meat product—preferably ham, sausage, and hamburger—, fishmeat, cake product, egg product—preferably seasoned egg rolls and eggcurd—, confectionery—preferably cookie, jelly, snacks, and chewing gum—,bread, noodles, pickle, smoked product, dried fish, seasoning.
 13. Thecomposition according to claim 11, wherein the binder microorganism orfragment thereof: is not a genetically modified microorganism as definedin Article 2(2) of Directive 2001/18/EC other than obtained through thetechniques of genetic modification listed in Annex 1B to said Directive2001/18/EC, and preferably even excluding microorganisms obtainedthrough the techniques of genetic modification listed in Annex 1B tosaid Directive 2001/18/EC, does not comprise a product resulting fromthe expression of genetic material heterologous to the order, preferablyto the family, even more preferably to the genus and most preferably tothe species of the binder microorganism. 14-15. (canceled)
 16. Themethod of claim 1, wherein: (a) the binder microorganism or fragmentthereof is used as a sensorically neutral anti-dental calculus agent,and wherein the composition is administered in an amount effective todelay or slow down formation of dental calculus in said human or animal;(b) the binder microorganism or fragment thereof is used as asensorically neutral anti-caries agent, and wherein the composition isadministered in an amount effective to delay or slow down formation ofcaries in said human or animal; and/or (c) the binder microorganism orfragment thereof is used as a sensorically neutral anti-oral malodoragent, and wherein the composition is administered in an amounteffective to reduceoral malodor in said human or animal.
 17. The methodof claim 1, wherein the binder microorganism is of familyLactobacillaceae, of genus Lactobacillus, Paralactobacillus, Pediococcusor Sharpea, of species Lactobacillus paracasei, Lactobacillus rhamnosus,Lactobacillus casei or Lactobacillus zeae, or is any of strains DSMZ16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ 16672 orDSMZ
 16673. 18. The method of claim 1, wherein the binder microorganismis in a viable, thermally inactivated or lyphilized form.
 19. The methodof claim 1, wherein the binder microorganism is in a thermallyinactivated form obtained by: treating in saturated steam at a pressureof 1-5 bar, 1-3 bar, or 2 bar; treating in saturated steam at a pressureof 2 bar at a temperature of 121° C. for 20 minutes; or freezing at −20°C. for at least 1 hour.
 20. The method of claim 1, wherein the bindermicroorganism or fragment thereof is capable of binding to at least onestrain, at least two strains, or at least three strains of mutansStreptococci selected from the group consisting of Streptococcus mutansserotype c (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923),Streptococcus mutans serotype f (NCTC 11060), Streptococcus sobrinusDSMZ 20742, Streptococcus ratti DSMZ 20564, Streptococcus cricetus DSMZ20562, Streptococcus ferus DSMZ 20646 and Streptococcus macacae DSMZ20714.
 21. The method of claim 1, wherein the binder microorganism orfragment thereof is not capable of binding to at least one, at leasttwo, at least three, or any microorganism selected from the groupconsisting of Streptococcus salivarius ssp. thermophilus, Streptococcusoralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralisDSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus sanguinisDSMZ
 20567. 22. The method of claim 1, wherein the binder microorganismis selected from the group consisting of: (i) microorganisms whichmetabolize D-lactose, but not L-sorbose and/or D-saccharose and/orD-inuline, (ii) microorganisms which metabolize inuline, (iii)microorganisms which metabolize L-sorbose, but not D-lactose and/orD-saccharose and/or inuline, and (iv) microorganisms which metabolizeL-sorbose, D-lactose and inuline, and preferably, wherein themicroorganism is selected from the group consisting of: (i)microorganisms which metabolize D-lactose, but not L-sorbose,D-saccharose and inuline, (ii) microorganisms which metabolizeL-sorbose, D-lactose and inuline, but not D-saccharose, (iii)microorganisms which metabolize L-sorbose, but not D-lactose,D-saccharose and inuline, and (iv) microorganisms which metabolizesL-sorbose, D-lactose, D-saccharose, but not inuline.